Apparatus for a mass production of monodisperse biodegradeable polymer-based microspheres and a multi-channel forming device incorporatable therein

ABSTRACT

Provided is an apparatus for a mass production of microspheres and a multichannel forming device incorporatable therein. The apparatus includes a multi-channel microsphere forming unit, a first source material reservoir containing the first source material and in fluid communication with the plurality of first microchannels, a second source material reservoir containing the second source material and in fluid communication with the plurality of second microchannels, a flow control unit configured to supply a first gas to the first source material reservoir at a first source material flow rate and to supply a second gas to a second source material reservoir at a second source material flow rate and a product reservoir for accommodating the microspheres formed from the multi-channel forming unit.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/788,906, filed Oct. 20, 2017, which claims benefit of U.S.Provisional Application 62/421,501 filed on Nov. 14, 2016, thedisclosures of which are incorporated herein by reference herein intheir entirety.

BACKGROUND

This invention generally relates to an apparatus and a method for massproducing monodisperse microspheres including therein biodegradablepolymers.

Pharmaceutical and bio-medical companies, in response to constantpressure to launch new products onto the market, are spending billionsof dollars annually on developing ever more complex and sophisticatedtherapeutics. However, it is widely acknowledged that many of thosetherapeutics never reach the market. Even the most promising compoundcan fail clinical trials if unfavorable pharmacokinetics or poordelivery prevents it from reaching its site of action. Controlling theparticulate characteristics of a drug formulation is an increasinglyimportant consideration in pharmaceutical manufacturing, for it canimprove a compound's probability of success by improving availabilityand reducing dosing.

For companies active in biological research, precise control overparticle characteristics enables development of novel and sophisticatedtherapies with advanced drug-delivery systems, and one of such advanceddrug-delivery systems currently being actively researched, developed andutilized is so called polymeric drug-delivery system(DDS), capable of,through the use of biodegradable, biocompatible and non-toxic polymers,such as, e.g., polylactic acid(PLA)/polyglycolic(PGA), providing acontrolled release of therapeutic agents in constant doses over longperiods, cyclic dosage, and tunable release of both hydrophilic andhydrophobic therapeutic agents(see FIG. 1).

One of the most well-known and representative medical products utilizingthe polymeric drug-delivery system is ELLANSE™ M from Aqutis/Sinclair, amedical product for tissue regeneration purposes. The product, i.e.,ELLANSE™ M, includes of monodisperse biocompatible and biodegradablepolymeric microspheres, PLA being the active ingredient thereof, and asa consequence of the polymer, i.e., PLA, biodegrading over 2 yearperiod, the tissue generation (or restoration or augmentation) effectsof the product end up lasting over the same period of time, providedthat the polymeric microspheres in the product are approximately of thesame size, i.e., monodispersed.

The most widely used methods for a mass production of polymeric DDS,include phase separation, spray drying and solventextraction-evaporation, as in the case of mass production of ELLANSE™ M.It is, however, almost impossible to control the size of the polymericmicrospheres using these processes, resulting in a wide particle sizedistribution, i.e., polydisperse. Since the optimum desired results ofthe product can be attained through a narrow microsphere sizedistribution or monodispersed, those microspheres of undesired sized,therefore, should be removed though a separate process, e.g., filtering,which, in turn, ends up, in addition to increasing the processing time,detrimentally affecting the final yield thereof.

SUMMARY

It is, therefore, an object of the present invention to provide anapparatus and a method for a large scale production of polymer-baseddrug delivery system based on microfluidics.

It is another object of the present invention to provide method foroptimizing the designing of an apparatus and processes for massproduction of monodisperse biodegradable polymer-based microspheres andbiodegradable polymer-based drug delivery systems.

It is still another object of the present invention to provide anapparatus and a method for a mass production of microspheresincorporating therein biodegradable polymers.

It is a further object of the present invention to provide an apparatusand a method for a high yielding mass production of monodispersebiodegradable polymer-based microspheres and biodegradable polymer-basedmedical products.

It is yet further object of the present invention to provide anapparatus and a method for a high yielding mass production ofmonodisperse biodegradable polymer-based microspheres and biodegradablepolymer-based medical products, the microspheres therein having adiameter ranging from 25 μm to 200 μm.

It is yet another object of the present invention to provide anapparatus and a method for a high yielding mass production ofmonodisperse biodegradable polymer-based microspheres and biodegradablepolymer-based medical products, capable of providing a controlledrelease of therapeutic drug/agent incorporated therein, the microspherestherein having a diameter ranging from 25 μm to 200 μm.

It is still another object of the present invention to provide anapparatus and a method for a mass production of microspheresincorporating therein a biodegradable polymer for use in medicalfillers, capable of providing a controlled biodegradation of thebiodegradable polymer.

It is still another object of the present invention to provide anapparatus and a method for a mass production of biodegradablepolymer-based microspheres incorporating therein a biodegradable polymerand a heartworm preventive drug/agent for use in heartworm preventives,capable providing a controlled release of the heartworm preventivedrug/agent.

It is still another object of the present invention to provide anapparatus and a method for a mass production of microspheresincorporating therein a biodegradable polymer and a hair loss preventivedrug/agent for use in hair loss preventives, capable of providing acontrolled release of the hair loss preventive drug/agent.

In accordance with one aspect of the present invention, there isdisclosed a method for design optimization of an apparatus for alarge-scale production of monodisperse microspheres and biodegradablepolymer-based drug delivery systems and process optimization usingtherewith, wherein the apparatus comprising a multichannel microsphereforming unit for initially generating the monodisperse microspheres andbiodegradable polymer-based drug systems of a desired diameter and shapeincluding therein at least two microchannels with a fixed dimension andat least two solutions, one solution known as a first solution and theother, a second solution, the first solution and the second solutionbeing immiscible with respect to each other, one of the solutionsincluding at least one biodegradable polymer dissolved therein in apredetermined amount, respectively known as a biodegradable polymerconcentration, and the other solution including at least one surfactantdissolved therein in a predetermined amount, respectively known as asurfactant concentration, each of the solutions flowing in therespective microchannel at a constant flow rate, known respectively as abiodegradable polymer solution flow rate and a surfactant solution flowrate, the microchannels flowing therein the biodegradable polymersolution and the surfactant solution merging with each other at amerging point at an angle, known respectively as the merging angle, toform an outflow microchannel, resulting in the formation of monodispersemicrospheres at the merging point having the desired diameter and shapeincluding therein the biodegradable polymers, the monodispersemicrospheres flowing out of the apparatus through the outflowmicrochannel along with the biodegradable polymer solution and thesurfactant solution, the formation of the monodisperse microsphereshaving the desired diameter and shape resulting from interaction of theimmiscibility of the solutions with respect to each other, the presenceof the surfactant and the concentration thereof in the surfactantsolution, the concentration of the biodegradable polymer in thebiodegradable polymer solution, the merging angle, wettability betweenthe solutions and the microchannel walls, the flow rate of thebiodegradable polymer solution and the surfactant solution and thedimension of the microchannels, the method involving an optimization ofthe factors mentioned above, the method further comprising:

-   -   (1) determining the biodegradable polymer to be used and a first        solvent in which the biodegradable polymer is to be dissolved,        resulting in the biodegradable polymer solution, and determine        the surfactant to be used and a second solvent in which the        surfactant is to be dissolved, resulting in the surfactant        solution, in such a way that the solutions are to be immiscible        with respect to each other;    -   (2) fixing a material on which the microchannels to be formed,        resulting in fixing of the wettability;    -   (3) fixing the diameter of the microspheres to be formed;    -   (4) fixing the dimension of the microchannels to be formed on        the material by determining a relationship between the dimension        and the diameter of the microspheres to be formed by forming the        microspheres by varying the channel dimension while holding        constant the flow rate of the biodegradable polymer solution,        the flow rate of the surfactant solution, the biodegradable        polymer concentration, the surfactant concentration and the        merging angle;    -   (5) forming the microchannels on the materials to be        incorporated in the multichannel microsphere forming unit in        such a way that each of the biodegradable polymer solutions and        the surfactant solutions flowing in the respective microchannels        to flow an identical distance from an inlet of the multichannel        microsphere forming unit to an outlet thereof, resulting in the        flow rates of the biodegradable polymer solution and the        surfactant solution within the respective microchannels in the        multichannel microsphere forming unit to remain constant        therethroughout;    -   (6) determining a relationship between the flow rate of the        surfactant solution on the diameter of the microspheres to be        formed by forming the microspheres by varying the flow rate of        the surfactant concentration while holding constant the channel        dimension, the flow rate of the biodegradable solution, the        biodegradable polymer concentration, the surfactant        concentration, the merging angle;    -   (7) determining a relationship between the flow rate of the        biodegradable polymer solution on the diameter of the        microspheres to be formed by forming the microspheres by varying        the flow rate of the biodegradable polymer concentration while        holding constant the channel dimension, the flow rate of the        surfactant solution, the biodegradable polymer concentration,        the surfactant concentration and the merging angle;    -   (8) determining a relationship between the surfactant        concentration on the diameter of the microspheres to be formed        by forming the microspheres by varying the surfactant        concentration while holding constant the channel dimension, the        flow rate of the surfactant solution, the flow rate of the        biodegradable polymer solution, the biodegradable polymer        concentration and the merging angle;    -   (9) determining a relationship between the biodegradable polymer        concentration on the diameter of the microspheres to be formed        by forming the microspheres by varying the surfactant        concentration while holding constant the channel dimension, the        flow rate of the surfactant solution, the flow rate of the        biodegradable polymer solution, the biodegradable polymer        concentration and the merging angle;    -   (10) determining a relationship between the merging angle on the        diameter of the microspheres to be formed by forming the        microspheres by varying the merging angle while holding constant        the channel dimension, the flow rate of the surfactant solution,        the flow rate of the biodegradable polymer solution, the        biodegradable polymer concentration and the surfactant        concentration; and    -   (11) determining the optimum design of the apparatus, including        therein the multichannel microsphere forming unit, for the large        scale production of the monodisperse microspheres and        biodegradable polymer-based drug delivery systems of the desired        diameter and shape incorporating therein the optimum merging        angle and the optimum dimension of the microchannels determined        through which the biodegradable polymer solution and the        surfactant solution flow and the optimum processes to be used        therewith incorporating therein the optimum flow rates of the        biodegradable polymer solution and the optimum concentrations of        the biodegradable polymer and surfactant in the respective        solutions determined.

In accordance with another aspect of the present invention, there isdisclosed an apparatus for a mass production of microspheres comprising:a multi-channel microsphere forming unit including a plurality of firstmicrochannels through which a first source material is flowable, aplurality of second microchannels through which a second source materialimmiscible with the first material is flowable, a plurality of firstmerging point where the plurality of first microchannels and theplurality of second microchannels are merged and the microspheresinitially get formed, and a plurality of third microchannels extendingfrom the plurality of first merging points and through which a firstmixed solution including therein the microspheres formed, the firstsource material and the second source material is flowable; a firstsource material reservoir containing the first source material and influid communication with the plurality of first microchannels; a secondsource material reservoir containing the second source material and influid communication with the plurality of second microchannels; a flowcontrol unit configured to supply a first gas to the first sourcematerial reservoir at a first source material flow rate and to supply asecond gas to a second source material reservoir at a second sourcematerial flow rate; and a product reservoir for accommodating themicrospheres formed from the multi-channel forming unit, wherein thefirst source material contained in the first source material reservoiris delivered to the plurality of first microchannels of themulti-channel forming unit in a flow rate corresponding to the firstsource material flow rate of the first gas, and the second sourcematerial contained in the second source material reservoir is deliveredto the plurality of second microchannels of the multi-channel formingunit in an flow rate corresponding to the second source material flowrate of the second gas, the first source material is delivered to theplurality of first microchannels at a constant first flow rate withoutfluctuation, and the second source material is delivered to theplurality of second microchannels at a constant second flow rate withoutfluctuation.

In accordance with yet another aspect of the present invention, there isdisclosed a multi-channel microsphere forming device for formingmicrospheres from a first source material and a second source materialimmiscible with the first source material, the device comprising: anupper case including a first annular manifold formed on a side of theupper case, a second annular manifold radially inside of the firstannular manifold on the side of the upper case, a first inlet lineconfigured to deliver the first source material and the second inletline configured to deliver the second source material to the secondannular manifold, a second annular manifold formed on the side of theupper case radially inside of the first annular manifold; a lower caseincluding a product exhausting hole formed at a center of the lowercase; a lower multi-channel plate disposed on the lower case andincluding a plurality of first microchannels radially arranged andformed on a side of the lower multi-channel plate, a plurality of secondmicrochannels radially arranged and formed on a side of the lowermulti-channel plate, a plurality of third microchannels radiallyarranged and formed on a side of the lower multi-channel plate, and acenter through-hole formed at a center of the lower multi-channel plate,wherein the plurality of first microchannels and the plurality of secondmicrochannels are merged at a plurality of first merging points and theplurality of third microchannels are arranged in direction to centerthrough-hole from the plurality of first merging points; and an uppermulti-channel plate disposed between the upper case and the lowermulti-channel plate, including a plurality of first channel connectionholes disposed between the plurality of first microchannels and thefirst annular manifold, and a plurality of second channel connectionholes disposed between the first annular manifold and the plurality ofsecond microchannels.

In accordance with still another object of the present invention, thereis disclosed a method for forming microspheres for use in medicalfillers, the method comprising: preparing a first source material inwhich a surfactant is dissolved in water; preparing a second sourcematerial in which the biodegradable polymer is dissolved in an organicsolvent; supplying the first source material and the second sourcematerial to multichannel forming unit in which the first source materialflows through a plurality of first microchannels, the second sourcematerial flows through the plurality of second microchannels, the firstsource material and the second source material are mixed with each otherat a plurality of merging points where the plurality of firstmicrochannels and the plurality of second microchannels meet with eachother, and a mixed solution of the first source material and the secondsource material flows through a plurality of third microchannelsextending from the plurality of first merging points; forming, in themixed solution, a plurality of microspheres including the biodegradablepolymer and having a diameter of 0.4 to 1.4 times the diameter of atarget size microspheres; and collecting a plurality of formedmicrospheres, wherein the width or height of the plurality of thirdmicrochannels is different from the diameter of the target sizemicrospheres differ by less than 30%.

In accordance with yet still another object of the present invention,there is disclosed a method for forming microspheres with abiodegradable and a heartworm preventive drug for use in heartwormpreventives, the method comprising: preparing a first source material inwhich a surfactant is dissolved in water; preparing a second sourcematerial in which the biodegradable polymer and the heartworm preventiveare dissolved in an organic solvent; supplying the first source materialand the second source material to multichannel forming unit in which thefirst source material flows through a plurality of first microchannels,the second source material flows through the plurality of secondmicrochannels, the first source material and the second source materialare mixed with each other at a plurality of merging points where theplurality of first microchannels and the plurality of secondmicrochannels meet with each other, and a mixed solution of the firstsource material and the second source material flows through a pluralityof third microchannels extending from the plurality of first mergingpoints; forming, in the mixed solution, a plurality of microspheresincluding the biodegradable polymer and the heartworm preventive drugand having a diameter of 0.4 to 1.4 times the diameter of a target sizemicrospheres; and collecting a plurality of formed microspheres, whereinthe width or height of the plurality of third microchannels is differentfrom the diameter of the target size microspheres by less than 30%.

It is still further another objective present invention, there isdisclosed a method for forming microspheres with a biodegradable polymerand hair loss preventive drug for use in a hair loss preventive, themethod comprising: preparing a first source material in which asurfactant is dissolved in water; preparing a second source material inwhich the biodegradable polymer and the hair loss preventive drug aredissolved in an organic solvent; supplying the first source material andthe second source material to multichannel forming unit in which thefirst source material flows through a plurality of first microchannels,the second source material flows through the plurality of secondmicrochannels, the first source material and the second source materialare mixed with each other at a plurality of merging points where theplurality of first microchannels and the plurality of secondmicrochannels meet with each other, and a mixed solution of the firstsource material and the second source material flows through a pluralityof third microchannels extending from the plurality of first mergingpoints; forming, in the mixed solution, a plurality of microspheresincluding the biodegradable polymer and the finasteride and having adiameter of 0.4 to 1.4 times the diameter of a target size microspheres;and collecting a plurality of formed microspheres, wherein the width orheight of the plurality of third microchannels is different from thediameter of the target size microspheres by less than 30%.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, not is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 illustrates a Biodegradable Microsphere loaded with Drug.

FIG. 2 is a Process Flow Chart & Apparatus Layout (Lab-scale).

FIG. 3 illustrates a Microchip Design viewed from the top (Photo).

FIG. 4 illustrates a Microchip Design (Top View).

FIG. 5 is a schematic diagram of the Microchip and the Flows establishedtherein.

FIG. 6 illustrates the effect of the Surfactant Concentration (weight %)on the Microsphere Diameter (μm).

FIG. 7 illustrates the effect of the Biodegradable Polymer Concentration(weight %) on the Microsphere Diameter (μm).

FIG. 8 is a photo of a Single Biodegradable Polymeric Microsphereobtained.

FIG. 9 is a photo of Monodisperse Biodegradable Polymeric Microspheresobtained.

FIG. 10 illustrates the effect of Microchannel Channel Dimension on theMicrosphere Diameter, with the Flow Rate of the Incoming Flows heldconstant.

FIG. 11 is a cross-sectional view of the Microchannel and theMicrosphere formed therein.

FIG. 12A illustrates the effect of the Flow Rate of the Water-PhaseSolution on the Microsphere Diameter (Flow Rate of the BiodegradablePolymer-phase Solution and the Dimension of the Microchannel Constant at100 μl/min and 200 μm*150 μm, respectively, while varying that of theWater-phase Solution).

FIG. 12B illustrates the effect of the Flow Rate of the Water-PhaseSolution on the Microsphere Diameter (Flow Rate of the BiodegradablePolymer-phase Solution and the Dimension of the Microchannel Constant at100 μl/min and 200 μm*150 μm, respectively, while varying that of theWater-phase Solution).

FIG. 12C illustrates the effect of the Flow Rate of the Water-PhaseSolution on the Microsphere Diameter (Flow Rate of the BiodegradablePolymer-phase Solution and the Dimension of the Microchannel Constant at100 μl/min and 200 μm*150 μm, respectively, while varying that of theWater-phase Solution).

FIG. 12D illustrates the effect of the Flow Rate of the Water-PhaseSolution on the Microsphere Diameter (Flow Rate of the BiodegradablePolymer-phase Solution and the Dimension of the Microchannel Constant at100 μl/min and 200 μm*150 μm, respectively, while varying that of theWater-phase Solution).

FIG. 12E illustrates the effect of the Flow Rate of the Water-PhaseSolution on the Microsphere Diameter (Flow Rate of the BiodegradablePolymer-phase Solution and the Dimension of the Microchannel Constant at100 μl/min and 200 μm*150 μm, respectively, while varying that of theWater-phase Solution).

FIG. 12F illustrates the effect of the Flow Rate of the Water-PhaseSolution on the Microsphere Diameter (Flow Rate of the BiodegradablePolymer-phase Solution and the Dimension of the Microchannel Constant at100 μl/min and 200 μm*150 μm, respectively, while varying that of theWater-phase Solution).

FIG. 13 illustrates the effect of the Flow Rate (μm/min) of theBiodegradable Polymer-phase Solution on the Microsphere Diameter (μm).

FIG. 14 is a block diagram of the Mass Production Apparatus forMicrospheres according to an embodiment of the present Invention.

FIG. 15A is a photo of a Prototype Microchip Developed for MassProduction of Microspheres (Top View).

FIG. 15B is a photo of a Prototype Microchip Developed for the MassProduction of Microspheres (Side View).

FIG. 15C is a photo of a Prototype Microchip Developed for the MassProduction of Microspheres (Side View).

FIG. 16 is a block diagram for describing the Flow Control Principle ofthe Flow Control Unit of the Mass Production Apparatus.

FIG. 17 is a graph illustrating the Flow Rate Control Principle of theFlow Rate Control Unit of the Mass Production Apparatus.

FIG. 18 is a block diagram illustrating the Channel-to-Channel FluidicConnection Relationship in the Multichannel Microsphere Forming Unit ofthe Mass Production Apparatus according to an exemplary embodiment ofthe present invention.

FIG. 19 is an assembled perspective view of the Multichannel Microsphereforming Unit of the Mass Production Apparatus according to an embodimentof the present invention.

FIG. 20 is an exploded perspective view of the Multichannel MicrosphereForming Unit of FIG. 19.

FIG. 21 is an exploded perspective view of the Multichannel Microsphereforming Unit of FIG. 19.

FIG. 22 is an assembled perspective front view of the MultichannelMicrosphere Forming Unit of FIG. 19;

FIG. 23 is a bottom view of the Upper Case of FIG. 19.

FIG. 24 is a top view of the Lower Case of FIG. 21 FIG. 25 is a top viewof the Upper Multichannel Plate of the Multichannel Microsphere FormingUnit according to an embodiment of the present invention.

FIG. 26 is a top view of the Lower Multichannel Plate of theMultichannel Microsphere Forming Unit according to an embodiment of thepresent invention.

FIG. 27 is a top view showing the Upper Multichannel Plate overlappedwith the Lower Multichannel Plate of the Multichannel MicrosphereForming Unit according to an embodiment of the present invention.

FIG. 28 is a top surface translucent diagram showing FIG. 27 with theFirst Annular Manifold and the Second Annular Manifold of the Upper Casein hidden line.

FIG. 29 is a cross-sectional view of the Upper Case, the UpperMultichannel Plate and the Lower Multichannel Plate taken along the lineX-X′ in FIG. 28.

FIG. 30 is a cross-sectional view of the Upper Case, the UpperMultichannel Plate and the Lower Multichannel Plate taken along the lineY-Y′ in FIG. 28.

FIG. 31 is a cross-sectional view of the Upper Case, the UpperMultichannel Plate and the Lower Multichannel Plate taken along the lineZ-Z′ in FIG. 28.

FIG. 32 is an exemplary schematic diagram illustrating the forming ofmicrosphere in the Mass Production Apparatus according to an embodimentof the present invention.

FIG. 33 is a block diagram of the Mass Production Apparatus according toanother embodiment of the present invention.

FIG. 34 is a block diagram illustrating a Channel-to-Channel FluidicConnection Relationship of the Multichannel Microsphere Forming Unit ofthe Mass Production Apparatus according to another embodiment of thepresent invention.

FIG. 35 is an assembled perspective view of the Multichannel MicrosphereForming Unit according to another embodiment of the present invention.

FIG. 36 is an exploded perspective view of the Multichannel MicrosphereForming Unit of FIG. 35.

FIG. 37 is an exploded perspective view of the Multichannel MicrosphereForming Unit of FIG. 35.

FIG. 38 is a bottom view of the Upper Case of the MultichannelMicrosphere Forming Unit of FIG. 35.

FIG. 39 is a top view of the Upper Multichannel Plate of theMultichannel Microsphere Forming Unit according to another embodiment ofthe present invention.

FIG. 40 is a top view showing the Upper Multichannel Plate overlappedwith the Lower Multichannel Plate of the Multichannel MicrosphereForming Unit according to another embodiment of the present invention.

FIG. 41 is a top surface translucent diagram showing FIG. 40 with theFirst Annular Manifold, the Second Annular Manifold and the ThirdAnnular Manifold of the Upper Case in hidden lines.

FIG. 42 is an exemplary schematic view showing the formation of theMulti-layered Microsphere MS2 in the Mass Production Apparatus accordingto another embodiment of the present invention.

FIGS. 43A to 43C illustrate forming of the multi-layered microsphere MS2as a consequence of MS1 coming in contact with the Third Material at theSecond Merging Point in accordance with another embodiment of thepresent invention.

FIGS. 44A to 44C illustrate forming of the multi-layered microsphere MS2as a consequence of MS1 coming in contact with the Third Materialintroduced relatively in large quantity at the Second Merging Point inaccordance with yet another embodiment of the present invention.

FIG. 45 is a block diagram of the Mass Production Apparatus according toyet another embodiment of the present invention.

FIG. 46 is an assembled perspective view of the Multichannel MicrosphereForming Unit of the Mass Production Apparatus in accordance with yetanother embodiment of the present invention.

FIG. 47 is an exploded perspective view of the Multichannel MicrosphereForming Unit shown in FIG. 46.

FIG. 48 is an exploded perspective front view of the MultichannelMicrosphere Forming Unit shown in FIG. 46.

FIG. 49 is an assembled perspective view of the Multichannel MicrosphereForming Unit shown in FIG. 46.

FIG. 50 is a bottom view of the Upper Case shown in FIG. 46.

FIG. 51 is a photographed image of the Microchannels around theProduct-exhausting Hole and an Enlarged exemplary partial view of theMicrochannels.

FIG. 52 is a lock diagram of the Mass Production apparatus incorporatingtherein a Channel-specific Valve Function according to yet anotherembodiment of the present invention.

FIG. 53 is an exemplary block diagram showing a Channel-to-ChanelFluidic Connection Relationship of the Valve Portion and theMultichannel Microsphere Forming Unit shown in FIG. 52 in accordancewith yet another embodiment of the present invention.

FIG. 54 is a bottom view of the Upper Case for implementing theMultichannel Microsphere Forming Unit shown in FIG. 53 according to yetanother embodiment of the present invention.

FIG. 55 is a block diagram of the Mass Production Apparatusincorporating therein a Buffer Tank according to still yet anotherembodiment of the present invention.

FIG. 56 illustrates an SEM Image Comparison of the Control Product(ELLASE™ M) and the Test Product (Perimore).

FIG. 57A to 57C illustrate a GPC Analysis of PCL (Polycaprolactone,PURAC®), the Control Product (ELLANSE™ M) and the Test Product(Perimore), respectively.

FIGS. 58A and 58B illustrate a Particle Size Distribution of the ControlProduct (ELLANSE™ M) and the Test Product (Perimore), respectively,obtained using a PSA(Particle Size Analyzer).

FIGS. 59A to 59D illustrate Storage Modulus, Loss Modulus, ComplexViscosity and Phase Angle of IVL-F_180530 IVL-F_180530, RESTYLANE®Perlane+Lido, AESTHEFILL® V200 and ELLANSE™ M, respectively, measuredand compared using a Rheometer (Kinexus, Malvern, U.K.).

FIGS. 60A and 60B illustrate an Injection Force Test for IVL-F_180530IVL-F_180530, RESTYLANE® Perlane+Lido, AESTHEFILL® V200 and ELLANSE™ Mobtained using TERUMO 27Gx3/4 and DN Co. 27G.

FIG. 61 illustrates SEM images illustrating the Change in Volume ofIVL-F_180530, RESTYLANE® Perlane+Lido, AESTJEFO::® V200 and ELLANSE™ Mafter being injected into laboratory mice.

FIG. 62 illustrates Chemical Formula/Structure of Moxidectin.

FIG. 63 illustrates a comparison of SEM Image Comparison of the ControlProduct (PROHEART SR-12) and the Test Product (DOP-02).

FIG. 64 illustrates a Size Distribution Analysis of the Control Product(PROHEART SR-12) and the Test Product (DOP-12).

FIGS. 65A and 65B illustrate a Release Test of the Control Product(PROHEART SR-12) and the Test Product (DOP-02).

FIGS. 66A to 66C illustrate SEM images of the Test Product (DOP-12)incorporating therein the biodegradable polymer (PLGA) at aconcentration of 40 weight %, 50 weight % and 60 weight %, respectively.

FIGS. 67A to 67C illustrate a Size Distribution Analysis of the TestProduct (DOP-12) incorporating therein the biodegradable polymer (PLGA)at a concentration of 40 weight %, 50 weight % and 60 weight %,respectively.

FIGS. 68A to 68C illustrate Release Tests of the Test Product (DOP-12)incorporating therein the biodegradable polymer (PLGA) at aconcentration of 40 weight %, 50 weight % and 60 weight %, respectively.

FIGS. 69A to 69C illustrate SEM images of the Test Product (DOP-12)incorporating Moxidectin and the biodegradable polymer (PLGA) at a ratioof 1:9, 1:6 and 1:4, respectively.

FIG. 70 illustrates Release Test Results (in-vitro) of the Test Product(DOP-12) incorporating therein Moxidectin and the biodegradable polymer(PLGA) at a ratio of 1:9, 1:6 and 1:4, respectively.

FIGS. 71A to 71C illustrate Release Test Results (laboratory animal) ofthe Test Product (DOP-2), the Control Product (PROHEARTSR-12) and aDirect Comparison of the Release Test Results of the Test Product andthe Control Product, respectively.

FIG. 72 illustrates the effect of Dutasteride and Finasteride onTestosterone.

DETAILED DESCRIPTION

This invention generally relates to an apparatus and a method for alarge scale production of microspheres incorporating thereinbiodegradable polymers; and more particularly to an apparatus and amethod for a high yielding large scale production of monodispersemicrospheres and biodegradable polymer-based medical products based onmicrofluidics which is a multidisciplinary field dealing with thebehavior, precise control and manipulation of fluids that aregeometrically constrained to typically a small scale, having one of thefollowing features:

-   -   (1) small volumes (μL, nL, μL, fL)    -   (2) small size    -   (3) low energy consumption    -   (4) effects of the micro domain

Droplet-based HCMMM (Highly Controlled Method for Mass-production ofMicrospheres) is a rapidly growing interdisciplinary field of researchcombining soft matter physics, biochemistry and microsystemsengineering, and is deemed to be a method having the distinction ofmanipulating discrete volumes of fluids in immiscible phases with lowReynolds number and laminar flow regimes. Interest in droplet-basedHCMMM systems has been growing substantially in past decades.Droplet-based HCMMM of the present invention offers the feasibility ofhandling miniature volumes of fluids conveniently, providing bettermixing and is deemed suitable for high throughput experiments/massproduction of polymeric DDS.

Currently, researches into drug delivery systems seeking to improve thepharmacological activity of therapeutic agents, i.e., activeingredients, by enhancing pharmacokinetics (absorption, distribution,metabolism and excretion) and also by amending pharmacodynamicproperties, such as the mechanism of action, pharmacological response,and affinity to the site of action are actively being carried out, andone of such a research involves the use of polymers, as (1) atherapeutic agent carrier to the site of action, in drug delivery, thetherapeutic agent being protected from interacting with other moleculeswhich could cause a change in the chemical structure of the activeingredient causing it to lose its pharmaceutical action; and (2) atherapeutic agent vehicle for providing a controlled release thereof(see FIG. 1 below). Moreover, polymeric carriers avoid the interactionof the therapeutic agent with macromolecules such as proteins, whichcould sequester the active ingredient preventing its arrival at theaction place.

If a polymer is to be used as a carrier, the next step is to design atype of polymeric structure that will permit obtaining the desiredrelease conditions. Therefore, the polymeric structure should be: i)biodegradable, because the chemical bonds that make up its chemicalstructure break; ii) disassemblable, because the various pieces formingthe polymer disassemble but the chemical bonds do not break; and iii)undisassemblable, because the chemical bonds do not disassemble orbreak, that is, the polymer remains unchanged. In the first two cases,micro-sized polymeric carriers could be used. However, if the polymericstructure of the polymer is neither biodegradable nor disassembable,then nano-sized polymers should preferably be used.

In the case of biodegradable polymers, another option to consider intheir design is the chemical structure of the polymer (degree ofhydrophobicity, covalent bonds between monomers, etc.), since the speedand degradation condition, and therefore, the rate and the site oftherapeutic agent release, can be modulated depending on the chemicalstructure of the polymer used.

An attempt was made by the inventor(s) to combine the ideas presentedhereinabove to develop an apparatus and a method for a largescale-production of biodegradable polymer-based monodispersemicrospheres.

Shown in FIG. 2 is the process flow chart and the layout of theapparatus for a lab-scale production of biodegradable polymer-basedmonodisperse microspheres based on HCMMM, respectively, wherein theprocess includes: (1) Preparing a biodegradable polymer-based solutionincluding therein the biodegradable polymer and a water-phase solutionincluding therein a surfactant; (2) Filtering the solutions prepared fora sterilization purpose; (3) Preparing biodegradable polymer-basedmicrospherical droplets by forcing the filtered solutions, i.e., thefiltered biodegradable polymer solution and the filtered water-phasesolution into a microchip (A in FIG. 3); (4) Sieving and filtering thebiodegradable polymer-based microspherical droplets; (5) Washing thesieved and filtered biodegradable polymer-based microspherical dropletswith pure water; (6) Filtering the biodegradable polymer-basedmicrospherical droplets washed with pure water; (7) Drying thebiodegradable polymer-based microspherical droplets; (8) Filtering thebiodegradable polymer-based microspherical droplets to obtain thebiodegradable polymer-based microspheres of a desired size, i.e.,monodisperse; (9) Mixing the biodegradable polymer-based microsphereswith a diluent that has been sterilized; (10) Charging and Sterilizingthe biodegradable polymer-based microspheres that have been mixed; and(11) Packaging the biodegradable polymer-based microspheres that havebeen charged and sterilized to obtain the final product. The steps (1)through (8) describe the method for producing monodisperse biodegradablepolymer-based microspheres, and the steps (9) though (11) can bechanged, modified or omitted depending on the final product desired. Thesteps shown in FIG. 2 are the steps required to produce medical fillersincluding therein monodisperse biodegradable polymer-based microsphereshaving a diameter of 50 μm, meaning that the specification of thefilters and the sieves shown in FIG. 2 can be changed depending on therequirements of the final product. Although steps (1) through (3) can beperformed in a clean room of class 10,000, steps (3) though (11) shouldbe performed in a clean room of class 100. The biodegradable polymer isdissolved in an oil-based solvent or organic solvent and the surfactant,in pure water, making the solutions prepared immiscible with respect toeach other. The solution including therein the biodegradable polymer andthe solution including therein the surfactant will, henceforth, be knownas the biodegradable polymer solution and the water-phase solution,respectively and the solutions, without saying, are immiscible, with therespect to one another.

The biodegradable polymers used in the present invention are selectedfrom the group consisting of: polylactic acid (PLA), polyglycolic acid(PGA), poly (lactic acid-glycolic acid), polycaprolactone and theirderivative groups, preferably polycaprolactone (PCL), but not limitedthereto. The number average molecular weight of the above biodegradablepolymers is not particularly limited, but ranges at between 5,000 and300,000, preferably between 8,000 and 250,000, and more preferablybetween 10,000 and 200,000.

The boiling point of the organic solvent used in the present inventionshould be less than 120° C. and is immiscible with water. For example,it is selected from the group consisting of: dichloromethane,chloroform, chloroethane, dichloroethane, trichloroethane and theirmixture groups, preferably dichloromethane, but not limited thereto.

The type of the surfactants used in the present invention is notparticularly limited, provided that it can facilitate a stableemulsification of the biodegradable polymer solutions. Specifically, itis selected from the group consisting of: nonionic surfactant, anionicsurfactant, cationic surfactant, and their mixture groups, morespecifically the group consisting of: methylcellulose,polyvinylpyrrolidon, lecithin, gelatin, polyvinyl alcohol,polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oilderivative, sodium lauryl sulfate, sodium stearate, ester amine, lineardiamine, fatty amine and their mixture groups, and preferably polyvinylalcohol (PVA), but not limited thereto.

One of the most critical importance issues in producing of monodispersebiodegradable polymer-based microsphere is the design of the microchipto be used, incorporating therein a plurality of microchannels based onHCMMM principles, the microchannels being the pathways through which thebiodegradable polymer-phase solution and the water-phase solutionprepared in the steps described hereinabove, and a solution includingtherein biodegradable polymer-based microspherical droplets formed flow,and FIGS. 3, 4 and 5 are a photo of the lab scale microchip used, aschematic diagram of the microchip viewed from the top and a simplifieddrawing at the crossing point of the microchannels therein,respectively. The microchip initially used in the present invention, asshown in FIGS. 3, 4 and 5, comprises three microchannels, i.e., 1, 2 and3 in FIG. 5, one of the microchannles being the pathway through whichthe biodegradable polymer solution flows, i.e., Flow 2, in FIG. 5 andthe remaining two microchannles, the pathway through which thewater-phase solution flow, i.e., Flow 1 and Flow 3 in FIG. 5, themicrochannel accommodating the flow of the biodegradable polymersolution being located between the microchannels accommodating the flowof the water-phase solution, the microchannels accommodating the flow ofthe water-phase solution merging with the microchannel accommodating theflow of the biodegradable polymer-phase solution at a merging point,i.e., 15, in FIG. 5, at an angle θ, the merging point being the locationwhere biodegradable polymer-based microspherical droplets get formed dueto the interaction of the solutions flowing in the microchannels and theimmiscibility of the solutions flowing through the microchannels meetingthereat, the microchannels accommodating the flows of the water-phasesolution having just an inlet, i.e., 11 and 13 in FIGS. 3, 4 and 5,respectively, through which the solution enters the microchannel and themicrochannel accommodating the flow of the biodegradable polymer-phasesolution having an inlet, i.e., 12, in FIGS. 3, 4 and 5, and an outlet,i.e., 14, in FIG. 5, the solution including the biodegradablepolymer-based microspherical droplets flowing out of the microchipthough the outlet, i.e., 14 in FIG. 5. The number of microchannels inthe microchip for accommodating the flows of the solutions can varydepending on the requirements of the final product. The solutionincluding the biodegradable polymer-based microspherical droplets willhenceforth be referred as the dispersed phase solution.

In the present invention, the water-phase solution enter the microchip Athrough the inlets 11 and 13, respectively, thereby establishing Flow 1and the Flow 3 in the microchannels 1 and 3, respectively, and meetsFlow 1 of the biodegradable polymer-phase solution at an angle between30° and 90° at the merging point 15, where the microspherical dropletsare formed due to the segmentation of the biodegradable polymersolution, i.e., Flow 2, by the water-phase solution, i.e., Flow 1 andFlow 3, and immiscibility of the two solutions, resulting in theformation of the dispersed phase solution, including therein themicrospherical droplets formed at the merging point 15, which flows outof the microchip through the outlet 14.

In the microchip of the present invention, the microchannels thereinwere formed using a deep reactive ion etching (DRIE) method, i.e., byetching, on a silicon wafer in a vertical direction and anodicallybonding glass thereon. The DRIE method is better suited than wet etchingin forming the microchannels, for it enables vertical etching and canalso provide a smooth surface when etching for 50 μm or deeper isrequired. Although, the microchannels of the present invention areformed on a silicon wafer, they can also be formed on glass, steel or onhydrophobic polymer wafer, such as PDMS. For example, the advantages ofusing these particular polymer surfaces are several. They are completelybioinert and antifouling, making them ideally suited forbiopharmaceutical processing and manufacturing. The wafer chipsthemselves are relatively inexpensive and completely disposable. What ismost important, the polymers enable establishment of segmented flowconditions with a high level of stability and reliability, allowing themass production of monodisperse biodegradable polymer-based microspherespossible.

It has been experimentally determined that if the water-phase solutionentering the microchip A through the inlets 11 and 13, respectively,thereby establishing Flow 1 and Flow 3 in the microchannels 1 and 3,respectively, meet the flow of the biodegradable polymer-phase solution,i.e., Flow 1 at an angle of either less than 30° or greater than at anangle 90°, it leads to widening of the size distribution of themicrospherical droplets formed, i.e., polydisperse microsphericaldroplets. Accordingly, to obtain microspherical droplets with a narrowsize distribution, i.e., monodisperse microspherical droplets, the angleat which the flows of the water phase solution merge with the flow ofthe biodegradable polymer solution, i.e., ø, should be set between 30°and 90°.

There are a number of parameters that need to be controlled in order toobtain the desired objective, and it has been determined through theexperiments by the inventor(s) that the critical/important parametersare the flow rate of the incoming flows, the viscosity of the incomingflows, the surfactant concentration in one of the incoming flows, thebiodegradable polymer concentration in the other incoming flow, thechannel wall wettability and the channel dimensions, and of thecritical/important parameters mentioned, it is possible to “fix” thechannel wall wettability by setting the materials to be used for themicrochip and the solutions for flowing through the microchannels andforming the microspherical droplets, and the viscosity, by fixing thefluids and the concentration of the biodegradable polymer and thesurfactant incorporating therein, respectively, leaving the flow rate ofthe incoming flows, i.e., incoming flow including therein thebiodegradable polymer and incoming flow(s) including therein thesurfactant, the channel dimension and the concentration of thebiodegradable polymers and the surfactant in the respective incomingflows, as the variables.

A series of experiments were performed to determine the effects of thevariables mentioned above, starting with the determination of theeffects of the concentration of the biodegradable polymers in thebiodegradable polymer-phase solution and the surfactant in thewater-phase solution, respectively.

In one embodiment of the present invention, the concentration of thesurfactant in the water-phase solution has been varied between 0.10 and0.50 weight % while holding the channel dimension and the incoming flowrates constant. In the surfactant concentration range specified, anincrease in the concentration leads to a corresponding decrease incoagulation of the microspheres formed, leading to a narrowparticle-size distribution due to a decrease in interfacial tension. Ifthe surfactant concentration (PVA) of the water-phase solutions fallsbelow 0.10 weight %, it results in widening particle-size distribution.However, if the concentration is 1.0 weight % or higher, it leads to adifficulty in washing and removing the surfactant from the microspheresobtained. It has further been experimentally determined that if theconcentration of the surfactant is equal to or greater than 0.15 weight%, the effect thereof on the microsphere diameter becomes negligible upto 0.25 weight %, flattening out at around 0.30 weight %, as shown inTable 1 and FIG. 6. The optimum/ideal/realistic surfactant concentrationin terms of the effectiveness and the cost factor, based on theexperimental results, is between 0.15 weight % and 0.30 weight %.

TABLE 1 Effect of the Surfactant Concentration (weight %) on theDiameter of Microsphere (μm) Surfactant Microsphere Concentration (wt %)Diameter (μm) 0.10 59.4 0.15 49.8 0.20 49.5 0.25 49.5 0.30 49.4

Another set of experiments were performed to determine the effects ofthe concentration of the biodegradable polymers in the biodegradablepolymer-phase solution by varying the concentration of the biodegradablepolymer in the biodegradable polymer-phase solution between 5 and 30weight % while holding the channel dimension and the flow rate of theincoming solutions constant. If the concentration of the biodegradablepolymer is less than 5 weight %, it results in microspheres not beingformed. If the concentration exceeds 30 weight %, it results inmicrospheres formed having non-spherical shape. There is shown in Table2 and FIG. 7 shows the effects of the concentration of the biodegradablepolymer (PCL) and the diameter of the microspheres formed.

Effect of the Biodegradable Polymer Concentration (weight %) on theMicrosphere Diameter (μm) PCL Concentration Microsphere Diameter (wt %)(μm) 10 53.6 15 49.8 20 49.5

According to the results summarized in Table 2 and shown in FIG. 6,there is roughly a linear relationship existing between theconcentration of the biodegradable polymer in the biodegradablepolymer-phase solution and the microsphere diameter, as defined by thefollowing relationship:

Microsphere Diameter=−0.408*(Biodegradable Polymer Concentration)+57.1with a standard deviation of 0.8048.

Based on the experimental results, the biodegradable polymerconcentration is between weight % and 30 weight %, and the relationshipdeveloped can be used to “fine tune” the microsphere diameter, if notfor all biodegradable polymers, at least for a PCL based system.

Photos of the microspheres obtained based on the experiments performedfor the embodiments above are shown in FIGS. 8 and 9.

Once the concentration of the surfactant and the biodegradable polymerget fixed in the respective incoming flows, i.e., within the rangestherefor specified above, the viscosity of the incoming flows willautomatically get fixed and once the material for the microchip getfixed, the channel wall wettability will also get fixed, since it isusually material dependent property, leaving the flow rate of theincoming flows and the microchannel dimension as thevariables/parameters to be determined/controlled.

In order to determine the effect of the microchannel dimension and theflow rate of the incoming flows on the microsphere diameter, threemicrochips with a microchannel having a channel dimension of 100 μm(width)×150 μm (depth), 200 μm (width)×150 μm (depth) and 300 μm(width)×150 μm (depth), respectively, were first obtained and thefollowing sets of experiments were performed therewith, i.e., holdingthe flow rate of water-phase solution with a fixed surfactantconcentration constant, i.e., at 0.25 weight %, while varying the flowrate of the biodegradable polymer solution with a fixed biodegradablepolymer concentration constant, i.e., at 15 weight %, using each of themicrochips and repeating the same procedure except for holding the flowrate of the biodegradable polymer-phase solution while varying the flowrate of the water-phase solution using each of the microchips (Pleaserefer to Table 3) and the results of these experiments are summarized inTable 4.

Tables 3: Experimental set-up to determine the Effect of Flow Rates andChannel Dimension on the Microsphere Diameter

Set 1: Channel Dimension: 300 μm×150 μm

Test 1: Varying the flow rate of the water-phase solution while holdingthe flow rate of the biodegradable polymer-phase solution constant at100 μl/min 1 2 3 PCL (conc.)% 15 15 15 PVA (conc.)% 0.25 0.25 0.25 PVA(conc.)%-reservoir 0.25 0.25 0.25 PCL (flow rate)μl/min 100 100 100 PVA(flow rate)μl/min 2000 3000 4000 Organic Solvent Extraction Temperaturetemp(° C.)-17° C. 1.5 1.5 1.5 temp(° C.)-20° C. 0.45 0.45 0.45 temp(°C.)-25° C. 0.45 0.45 0.45

Test 2: Varying the flow rate of the water-phase solution while holdingthe flow rate of the biodegradable polymer-phase solution constant at130 μl/min 1 2 3 PCL (conc.) % 15 15 15 PVA (conc.) % 0.25 0.25 0.25 PVA(conc.) %—reservoir 0.25 0.25 0.25 PCL (flow rate) μl/min 130 130 130PVA (flow rate) μl/min 2000 3000 4000

Test 3: Varying the flow rate of the water-phase solution while holdingthe flow rate of the biodegradable polymer-phase solution constant at160 μl/min 1 2 3 PCL (conc.) % 15 15 15 PVA (conc.) % 0.25 0.25 0.25 PVA(conc.) %—reservoir 0.25 0.25 0.25 PCL (flow rate) μl/min 160 160 160PVA (flow rate) μl/min 2000 3000 4000Set 2: Channel Dimension: 200 μm×150 μm

Test 1: Varying the flow rate of the water-phase solution while holdingthe flow rate of the biodegradable polymer-phase solution constant at100 μl/min 1 2 3 PCL (conc.) % 15 15 15 PVA (conc.) % 0.25 0.25 0.25 PVA(conc.) %—reservoir 0.25 0.25 0.25 PCL (flow rate) μl/min 100 100 100PVA (flow rate) μl/min 2000 3000 4000 Organic Solvent ExtractionTemperature temp(° C.)—17° C. 1.5 1.5 1.5 temp(° C.)—20° C. 0.45 0.450.45 temp(° C.)—25° C. 0.45 0.45 0.45

Test 2: Varying the flow rate of the water-phase solution while holdingthe flow rate of the biodegradable polymer-phase solution constant at130 μl/min 1 2 3 PCL (conc.) % 15 15 15 PVA (conc.) % 0.25 0.25 0.25 PVA(conc.) %—reservoir 0.25 0.25 0.25 PCL (flow rate) μl/min 130 130 130PVA (flow rate) μl/min 2000 3000 4000

Test 3: Varying the flow rate of the water-phase solution while holdingthe flow rate of the biodegradable polymer-phase solution constant at160 μl/min 1 2 3 PCL (conc.) % 15 15 15 PVA (conc.) % 0.25 0.25 0.25 PVA(conc.) %—reservoir 0.25 0.25 0.25 PCL (flow rate) μl/min 160 160 160PVA (flow rate) μl/min 2000 3000 4000

TABLE 4 Effect of Microchannel Dimension and Flow Rate of Incoming Flowson Microsphere Diameter Flow Rate of Flow Rate of Biodegradable Water-Polymer-phase phase Microchannel Solution: Solution: MicrosphereDimension 15 wt % PCL 0.25 wt % PVA Diameter (μm*μm) (μl/min) (μl/min)(μm) 200*150 100 2000 146.7 200*150 100 3000 125.2 200*150 100 400097.67 200*150 130 2000 128 200*150 130 3000 60.87 200*150 130 4000 67.09200*150 160 2000 164.4 200*150 160 3000 160.2 200*150 160 4000 66.04300*150 100 2000 213.9 300*150 100 3000 145.1 300*150 100 4000 173.4300*150 130 2000 230.6 300*150 130 3000 134.9 300*150 130 4000 167.8300*150 160 2000 268.1 300*150 160 3000 234.1 300*150 160 4000 228

Effects of the Microchannel Dimension and Flow Rate of the IncomingFlows on the Microsphere Diameter

Based on the experimental results shown in Table 4, effects of themicrochannel dimension and the flow rate of the incoming flows on thediameter of the microspheres formed, the microchannel dimension has moreimmediate/direct/controllable effects on the diameter/size of themicrospheres formed than the flow rate of the incoming flows, and theeffects of each factors, i.e., the dimension and the flow rate of theincoming flows, will be described in detail hereinbelow.

Effect of the Microchannel Dimension on the Diameter of the MicrospheresDetermined while Holding the Flow Rate of the Incoming Flows Constant

As shown in FIG. 10, there is a strong correlation, i.e., usually alinear relationship or close thereto, between the channel dimension ofthe microchannel and the microsphere diameter, d′: An increase inchannel dimension results in a corresponding increase in microspherediameter, d′. Ideally speaking, the cross-section of the microchannelshould be a circle, i.e., similar to the cross-section of themicrosphere to be formed, which is an extremely difficult task, andaccordingly, the next best solution thereto would be formation ofmicrochannels having a cross-section thereof being a square, as shown inFIG. 11, i.e., the depth (d) and the width (w) of the cross-section ofthe microchannels being identical. It has been experimentally determinedthat in order to expedite an efficient formation of monodispersemicrospheres, the dimension of the microchannel, i.e., the width (w) andthe depth (d), as shown in FIG. 11, should be within 30% of the desireddiameter, d′. That is, if the desired microsphere diameter is 100 μm,the width (w) or the depth (d) of the microchannel should preferably bewithin 70 μm and 130 μm or a combination thereof. Another way of sayingwhat has been stated above is that the cross-sectional area of themicrochannels, i.e., depth (d)×width (w), should be within 30% of thecross-sectional area of the microsphere desired, i.e., (0.5 d′)×(0.5d′)π. Should this task of forming microchannels whose cross-section is asquare is difficult, it has been experimentally further determined thatat least either one of the depth (d) or the width (w) of the crosssection of the microchannels should be within 30% of the desireddiameter of the microspheres to be formed.

Effect of the Flow Rate of Incoming Flows on the Microsphere Diameter

According to the results obtained, of the two flow rates, i.e., the flowrate of the water-phase solution including therein the surfactant andthat of the biodegradable polymer-phase solution including therein thebiodegradable polymer, the flow rate of the water-phase solution is morecritical than that of the biodegradable polymer-phase solution, althoughnot as critical as that of the microchannel dimension, as can be seen inFIG. 10, in affecting the diameter of the microspheres being formed, forthe flow of the water-phase solution, and hence the rate thereof, is theflow providing the force for segmenting the biodegradable polymer-phasesolution to the desired size of microspheres, i.e., the microsphereshaving the desired diameter, and the effects of the flow rate of thewater-phase solution are shown in Table 5 and FIGS. 12A through 12F. Ascan be seen from Table 5 and FIGS. 12A through 12F, the diameter of themicrospheres is generally inversely linearly proportional to the flowrate of the water-phase solution, i.e., an increase in the flow rateresults in a corresponding decrease in the diameter of the microspheres,and this relationship only is applicable only in a certain range of theflow rate, for if the flow rate of the water-phase solutions is too low,it results in the formation of biodegradable polymer-based microsphereshaving a wide microsphere diameter distribution and if the flow rate ismore too high, it results in the formation of microspheres havingdiameters of less than 10 μm diameter, possibly resulting in thebiodegradable polymer-based microspheres being easily absorbed by thebody or the biodegradable polymer-based microspheres having a too shortof biodegradation time when injected into the body. At the crossingpoint, the relatively slower moving biodegradable polymer-phase solutiongets pressed by the relatively faster moving water-phase solution,resulting disrupting the flow of the biodegradable polymer-phasesolution, isolating a small amount of the biodegradable polymer-phasesolution to be surrounded by the water-phase solution, leading to theformation of microspherical droplets due to the surface tension. For thecurrent system involving a microchannel made up of silicon wafer, awater-phase solution including therein PVA as the surfactant and abiodegradable polymer-phase solution including therein PCL as thebiodegradable polymer, the range of the flow rate of the water-phasesolution that be incorporated is between 500 μl/min and 5000 μl/min.

TABLE 5 Effect of the Flow Rate of the Water-phase Solution on theDiameter of the Microspheres (a) PCL PVA Microsphere Diameter (μl/min)(μl/min) (μm) 200 (μm) × 150 (μm) 100 2000 146.7 100 3000 125.2 100 400097.7 (b) PCL PVA Microsphere Diameter (μl/min) (μl/min) (μm) 200 (μm) ×150 (μm) 130 2000 128 130 3000 60.8 130 4000 67.1 (c) PCL PVAMicrosphere Diameter (μl/min) (μl/min) (μm) 200 (μm) × 150 (μm) 160 2000164.4 160 3000 160.2 160 4000 66.0 (d) PCL PVA Microsphere Diameter(μl/min) (μl/min) (μm) 300 (μm) × 150 (μm) 100 2000 213.9 100 3000 145.1100 4000 173.4 (e) PCL PVA Microsphere Diameter (μl/min) (μl/min) (μm)300 (μm) × 150 (μm) 130 2000 230.6 130 3000 134.9 130 4000 167.8 (f) PCLPVA Microsphere Diameter (μl/min) (μl/min) (μm) 300 (μm) × 150 (μm) 1602000 268.1 160 3000 234.1 160 4000 228.0

Effect of the Flow Rate of the Biodegradable Polymer-Phase Solution onthe Microsphere Diameter

In another embodiment of the present invention, the flow rate of thebiodegradable polymer-phase solution ranges between 10 μl/min and 500μl/min, and preferably between 50 μl/min and 200 μl/min. If thebiodegradable polymer-phase solution flows at a rate lower 50 μl/min, itleads to microspheres becoming smaller, resulting in shortening of thebiodegradation time and having a wide particle-size distribution.Meanwhile, if the biodegradable polymer-phase solution flow at a ratehigher than 200 μl/min, it leads to microspheres becoming larger, makingit difficult to inject and having a wide particle-size distribution.There is shown in Table 6 and FIG. 13 the effects of the flow rate ofthe biodegradable polymer-phase solution on the diameter of themicrospheres formed.

According to the results shown in Table 6 and FIG. 13, there is a stronglinear relationship between the flow rate and the diameter, as definedby the following relationship:

Microsphere Diameter=0.095*(Flow Rate)+38.387 with a standard deviationof 0.9999.

TABLE 6 Effect of the Flow Rate (μl/min) of the Biodegradable PolymerSolution on the Microsphere Diameter (μm) PCL Flow Rate MicrosphereDiameter (μl/min) (μm) 120 49.8 140 51.7 160 53.6

Although there is a linear relationship between the flow rate of thebiodegradable polymer-phase solution and the diameter of themicrospheres formed, the effect thereof on the diameter is not ascritical as those of the channel dimension and the flow rate of thewater-phase solution, i.e., very minor effect on the diameter, and henceit should be used as a “controlling parameter for fine-tuning” thediameter of the final microspheres formed, i.e., after using the channeldimension and the flow rate of the water-phase solution to initially fixthe diameter of the microspheres obtained.

It has been experimentally determined that the ratio of the flow rate ofthe biodegradable polymer-phase solution to the flow rate of thewater-phase solution ranges between 1:2 to 1 to 100, preferably between1:2 to 1:50.

Hereinafter, the present invention will be described in further detailthrough the following example, using a microchip incorporating thereinthe simplest form of microchannles, i.e., lab-scale, shown in FIGS. 3, 4and 5 and the principles developed hereinabove. The example is only forillustrating the invention more specifically, and it is obvious to thepersons in the industry with common knowledge that the scope of theinvention is not limited to the examples according to the content of theinvention.

Firstly, 10 mL of the biodegradable polymer-phase solution was obtainedby dissolving polycaprolactone (PCL) with Mn ˜45,000 or less, wherein Mnis the number average molecular weight obtained by dividing themolecular weight of the molecular species comprising polymeric compoundswith molecular weight distribution by the number or mole fraction, in asolvent, e.g., dichloromethane (solvent, melting point: 39.6° C.) at aconcentration of 15 weight %. Secondly, 250 mL of the water-phasesolution is prepared by dissolving a surfactant, i.e., polyvinyl alcohol(PVA) having a molecular weight of 85000 to 124000, in purified water ata concentration of 0.25 weight %. Thirdly, 100 mL of the receiversolution is prepared by dissolving polyvinyl alcohol in purified waterat a concentration of 0.25 weight percent. The above biodegradablepolymer-phase solution is injected into one of the microchannels, i.e.,Channel 2, at a fixed flow rate, e.g., 100 μl/min′ as Flow 2 and theprepared water-phase solution at 90° angle from the flow of thebiodegradable polymer solution through Channel 1 and Channel 3, as Flow1 and Flow 3 at a fixed rate, e.g., 1000 μl/min.

Dispersed phase is generated at the merging point 15 of thebiodegradable polymer-phase solution and the water phase solution.Thereafter, the dispersed phase, including therein the microsphericdroplets formed at the merging point 15, flows out through the outlet 14to be collected in the receiver solution, and dichloromethane solvent isextracted therefrom by keeping the receiver solution included thereinthe dispersed phase at room temperature (25° C.) for about 24 hours,resulting a water-phase solution containing biodegradable polymer-basedmicrospherical droplets. Microspherical droplets are separated out fromthe water phase solution through a filtering process. Microspherescontaining biodegradable polymers are finally obtained by washing themicrosphere droplets to remove the remaining polyvinyl alcohol anddichloromethane solution, followed by a drying process (Please refer toFIG. 2).

To be more specific, the method described above is largely composed offour flows, i.e., Flow 1, Flow 2, Flow 3 and Flow 4: Flow 2 establishedby the flow of biodegradable polymer-phase solution prepared bydissolving biodegradable polymer in an organic solvent; Flow 1 and Flow3, established by the flow of the water-phase solutions prepared bydissolving the surfactant in purified water; and Flow 4, established bythe flow resulting from Flows 1, 2 and 3 meeting at the merging point15. Flow 2 runs in the same direction as Flow 4, and Flow 1 and Flow 3run toward each other and merge together at the crossing point 15(Please refer to FIG. 5). The flow rate of Flow 1, Flow 2 and Flow 3 andthe angle at which Flow 1 and Flow 3 merge with Flow 2 affect the sizeof microspheres generated, the particle size distribution and theproduction yield.

In the present invention, those biodegradable polymer-basedmicrospherical droplets formed at the merging point 15 are collected ina receiver containing a receiver solution including therein asurfactant, and the reason for using the receiver solution includingtherein the surfactant is to prevent those biodegradable polymer-basedmicrospherical droplets from coagulating.

Those biodegradable polymer-based microspherical droplets collected inthe step described above undergo a first drying at a temperature between0° C. and 50° C., and preferably between, 20° C. and 25° C. When anemulsion in form of a droplet is kept at under the boiling point of theorganic solvent for a certain amount of time, for example, between 12and 48 hours, the organic solvent therein gets extracted from thedroplet, resulting in the formation of a microsphere thoughsolidification process.

The biodegradable polymer-based microspheres formed in the stepdescribed above are filtered and are then washed with purified water atleast once, preferably between one and three times to remove theremaining surfactant and solvent, followed by one more filteringprocess. The washing process may be repeated until the remainingsurfactant and the solvent are completely removed.

In an embodiment herein, the step described above is followed by anotherdrying process and the drying method to be used is not particularlylimited. Although, however, the drying method to be used is notparticularly limited, it is preferred that the microspheres should bedried in vacuum or using lyophilization to minimize heat damages to thebiodegradable polymer included in the microspheres.

The average diameter of the biodegradable polymer-based microspheresproduced using the apparatus and method prescribed hereinabove rangesbetween 10 μm and 200 μm, preferably between 30 μm and 150 μm. If theaverage diameter of the microspheres is less than 10 μm, themicrospheres may easily end getting absorbed in the body when injectedor the biodegradation time thereof may end of being too short to fulfilltheir intended purpose. On the other hand, if the average diameter ofthe microspheres exceeds 150 μm, it may result in creating difficultiesin interdermal injection thereof.

When microspheres are prepared using a conventional batch process, forexample, solvent extraction-evaporation process, it may lead to theformation of microspheres having irregular particle sizes and arelatively wider particle-size distribution. In such a case, themicrospheres of an undesired size is separated out through, for example,filtering or sieving, to obtain the monodispersity of the microspheres,which will, in turn, end up detrimentally affecting the final yield ofthe process, in addition to unnecessarily complicating the manufacturingprocess. However, using the apparatus and the method based onimmiscibility of the solutions used and basic underlying principlesbehind HCMMM, described herein above, it is possible to manufacturemonodisperse biodegradable polymer-based microspheres, with a higheryield and enhanced simplicity and controllability.

Biodegradable polymer-based microspheres prepared in accordance with theapparatus and the method of the present invention is not limited to aparticular usage. For example, they may be used in skin aesthetics or asa medical filler requiring bioresorption, particularly as injectablesubcutaneous or intradermal fillers that can be implanted into the body,but not limited only thereto.

Although the reabsorbability time of biodegradable polymers-basedmicrospheres prepared in accordance with the apparatus and the method ofthe present invention is not particularly limited, it should preferablybe between one and three years, considering that they are used asbiodegradable fillers for skin aesthetics or medical purposes.

Summary of the Experimental Results

To summarize there are five important parameters to be controlled tooptimize the mass production of monodisperse microspheres, namely, theflow rate of the incoming flows, the viscosity of the incoming flow, thesurfactant concentration in the water-phase solution, the biodegradablepolymer concentration in the biodegradable polymer-phase solution, thechannel wall wettability and the channel dimensions, and of thecritical/important parameters mentioned, it is possible to fix thechannel wall wettability by setting the material to be used for themicrochip, and the viscosity, by fixing the fluids and the concentrationof the biodegradable polymer and the surfactant being incorporatetherein, respectively, the optimal concentration thereof being easilydetermined through experiments, leaving the flow rate of the incomingflows, i.e., incoming flow including therein the biodegradable polymerand incoming flow(s) including therein the surfactant, the channeldimension and the concentration of the surfactant and the biodegradablepolymer in the respective incoming flows as the key variables to becontrolled.

It has been experimentally determined that it the concentration of thesurfactant is equal to or greater than 0.15 weight %, the effect thereofon the microsphere diameter becomes negligible up to 0.25 weight %,flattening out at around 0.30 weight %.

As for the effect of the concentration of the biodegradable polymer, ifthe concentration of the biodegradable polymer is less than 5 weight %,it results in microspheres not being formed. If the concentrationexceeds 30 weight %, it results in microspheres formed havingnon-spherical shape, and for those concentrations between 5% and 30%,there is a strong correlation, i.e., for example, roughly a linearrelationship, existing between the concentration of the biodegradablepolymer in the biodegradable polymer-phase solution and the microspherediameter, as defined by the following relationship:

Microsphere Diameter=−0.408*(Biodegradable Polymer Concentration)+57.1with a standard deviation of 0.8048.

Based on the experimental results, the biodegradable polymerconcentration is between weight % and 30 weight %, and the relationshipdeveloped can be used to fine tune the microsphere diameter, if not forall biodegradable polymers, at least for the PCL based system.

Of the remaining variables, i.e., the channel dimension and the flowrate of the incoming flows, it has been determined that the microchanneldimension has the most immediate/direct/controllable effects on thediameter/size of the microspheres formed than the flow rate of theincoming flows. It has been experimentally determined that there is astrong correlation, i.e., usually a linear relationship or closethereto, between the channel dimension of the microchannel and themicrosphere diameter: An increase in channel dimension results in acorresponding increase in microsphere diameter. Ideally speaking, thecross-section of the microchannel should be a circle, i.e., similar tothe cross-section of the microsphere to be formed, which is an extremelydifficult task, and accordingly, the next best solution thereto would beformation of microchannels having a cross-section thereof being asquare, i.e., the depth (d) and the width (w) of the cross-section ofthe microchannels being identical. It has been experimentally determinedthat in order to expedite an efficient formation of monodispersemicrospheres, the dimension of the microchannel, i.e., the width (w) andthe depth (d), should be within 30% of the desired diameter of themicrospheres to be produced, i.e., the cross-sectional area of themicrochannels, should be within 30% of the cross-sectional area of themicrospheres desired. Should this task of forming microchannels whosecross-section is a square is found to be difficult, it has beenexperimentally further determined that at least either one of the depth(d) or the width (w) of the cross section of the microchannels should bewithin 30% of the desired diameter of the microspheres to be formed.

According to the results obtained, of the two flow rates, i.e., the flowrate of the water-phase solution including therein the surfactant andthat of the biodegradable polymer-phase solution including therein thebiodegradable polymer, the flow rate of the water-phase solution is morecritical than that of the biodegradable polymer-phase solution inaffecting the diameter of the microspheres being formed, for the flow ofthe water-phase solution, and hence the rate thereof, is the flowproviding the force for segmenting the biodegradable polymer-phasesolution to the desired size of microspheres, i.e., the microsphereshaving the desired diameter, for the diameter of the microspheres isgenerally inversely linearly proportional to the flow rate of thewater-phase solution, i.e., an increase in the flow rate results in acorresponding decrease in the diameter of the microspheres, and thisrelationship only is applicable only in a certain range of the flowrate.

Although not as critical as the flow rate of the water-phase solution inaffecting the microsphere diameter, there is a strong correlationbetween the flow rate of the biodegradable polymer-phase solution andthe microsphere diameter, i.e., usually a linear relationship, in aspecific range of flow rates and hence, can be used as a controllingparameter for fine-tuning the diameter of the microspheres to be formedafter using the channel dimension and the flow rate of the water-phasesolution to initially fix the diameter of the microspheres obtained.

It has been experimentally determined that the ratio of the flow rate ofthe biodegradable polymer-phase solution to the flow rate of thewater-phase solution ranges between 1:2 to 1 to 100, preferably between1:2 to 1:50.

Further, to obtain microspherical droplets with a narrow sizedistribution, i.e., monodisperse microspherical droplets, the angle atwhich the flows of the water-phase solution merge with the flow of thebiodegradable polymer-phase solution should be set between 30° and 90°.

Mass Production

An optimized appratus and process for a mass production of monodispersemicrosperes has been realized based on the the results obtained anddecribed hereinabove. There are illustrated in FIG. 14 and FIGS. 15A toC the lay-out of the apparatus for mass production and the photos of aprototype microchip to be incorporated into Multi-Channel MicrosphereForming Unit 100 in FIG. 14. Theoretically, the easiest way to developan apparatus for mass production of microspheres is to developmicrochips incorporating therein a plurality of single microsphereproduction units shown in FIG. 5 in parrallel and, in turn, arrangethose microchips in parrallel. As an example of such a microchip, thereare shown in FIGS. 15A through 15C photos of a prototype microchipdeveloped for the mass production of microspheres incorporating thereinseven such microsphere production units shown in FIG. 5 in a singlemicrochip. To achieve a mass production of microspheres using themicrochips shown in FIGS. 15A to C, they are arranged in parrallel, thetotal number of microchips needed depending on the mass productionrequirement.

In the microchip shown in FIG. 15A to C, a plurality of microsphereproduction units are formed on hydrophobic polymer wafer, such as PDMS,for the advantages of using these particular polymer surfaces areseveral. They are completely bio-inert and antifouling, making themideally suited to biopharmaceutical processing and manufacturing. Thewafer chips themselves are relatively inexpensive and completelydisposable. What is most important, the polymers enable establishment ofsegmented flow conditions with a high level of stability andreliability, making the mass production of biodegradable polymer-basedmicrospheres possible.

However, the use of such a large number of microsphere productions unitsin the Multichannel Microsphere forming Unit, and hence thecorresponding number of microchannels associated therewith, neccesitatea need for the supply of corresponding controlled amount of fluidflowing thereinto and this is an extremely difficult task usingconventional lab-scale fluid supply units, e.g., a fluid pump, for theconventional lab-scale fluid supply units, in general, have a pressurefluctuation corresponding to the operating cycle thereof. Accordingly, asolution is found capable of providing a constant flow condition withineach microchannel with a constant pressure therewithin, i.e.,consistency in the flow rate between the microchannels.

In addition, an increased number of microchannels in the microchip,i.e., an increased in microchannel density within the microchip, resultsin the microchannel structure therein becoming very complicated/complex,resulting in flow paths therein becoming inconsistent, which, in turn,causing the flow resistance within each microchannel becominginconsistent with respect to one another, and if the flow resistancesare different for each microchannel, the flow within each microchannelwill end up being different. Accordingly, in order for the microchipdescribed hereinabove to be used in mass production of microspheres, ameans capable of providing a consistent flow condition, and hence, aneven flow rate, in each of the microchannels are determined, prior tothe microchip being used in mass production of microspheres.

In order to solve the above-mentioned problems, new concepts capable ofproviding and maintaining a constant and equal flow within each of themicrochannels have been proposed and developed. This concept may beimplemented as an exemplary embodiment, and some of the exemplaryembodiments thereof are described below.

Referring to FIG. 14, the mass producing apparatus for microspheresaccording to an embodiment of the present invention comprises a firstmaterial reservoir 300, a second material reservoir 400, a flow ratecontrol unit 200, a multichannel microsphere forming unit 100, a productreservoir 500 and a dispersion reservoir 700.

The first material reservoir 300 comprises a first raw material in theform of a water-phase solution comprising pure water dissolved therein asurfactant, for example, 0.25% by weight of polyvinyl alcohol(PVA)having a molecular weight of 8500 to 124000 as the surfactant dissovedin pure water.

The first raw material is sterilized through a sterilization process,for example, by being passed through a sterilization filtration filter.The filtered first raw material is then introduced into the firstmaterial reservoir 300 through a first material inlet 310. After thesterilized first raw material is sufficiently or completely introducedinto the first material reservoir 300, the first material inlet valve312 mounted in the first material inlet 310 is shut off, therebyisolating the the first material reservoir 300 from the outside and thesterilization state to be maintained therewithin.

The second material reservoir 400 comprises a second raw material thatis an oil-phase solution comprising an organic solvent and abiodegradable polymer dissoved therein. For example, the organic solventof the second raw material may be dichloromethane(solvent, melting point39.6° C.), and the biodegradable polymer may be polycaprolactone(PCL)with Mn of about 45,000 or less in dichloromethane at a concentration of15 wt %, wherein Mn is the number average molecular weight obtained bydividing the molecular weight of the molecular species comprisingpolymeric compounds with molecular weight distribution by the number ormole fraction.

The second raw material is sterilized through a sterilization process,for example, by being passed through a sterilization filtration filter.The sterilized second raw material is introduced into the secondmaterial reservoir 400 through a second material inlet 410. After thesterilized second raw material is sufficiently or completely introducedinto the second material reservoir 400, the second material inlet valve412 installed in the second material inlet 410 is shut off, therebyisolating the second material reservoir 400 from the outside and thesterilization state to be maintained therewithin.

The first raw material stored in the first material reservoir 300 andthe second raw material stored in the second material reservoir 400 arethen transferred to the multichannel microsphere forming unit 100through the first material outlet 320 and the second raw material outlet420, respectively.

The flow rate control unit 200 is in fluid communication with the firstmaterial reservoir 300 through the first flow control line 210 and influid communication with the second material reservoir 400 through thesecond flow control line 220, respectively, the flow control unit 200introducing a first gas having a first raw material flow rate into thefirst material reservoir 300 and a second gas having a second rawmaterial flow rate into the second material reservoir 400. The first gasand the second gas may be of substantially the same kind of gas, forexample, clean air or an inert gas.

The first raw material stored in the first material reservoir 300 isdelivered to the multichannel microsphere forming unit 100 in an amountcorresponding to the first raw material flow rate of the introducedfirst gas. Similarly, the second raw material stored in the secondmaterial reservoir 400 may be delivered to the multichannel microsphereforming unit 100 in an amount corresponding to the second raw materialflow rate.

FIG. 14 is Block Diagram of a Mass Production Apparatus for Microspheresaccording to an Embodiment of the Present Invention. FIG. 15A is Photoof a Prototype Microchip Developed for Mass Production of Microspheres(Top View). FIG. 15B is Photo of a Prototype Microchip Developed forMass Production of Microspheres (Side View). FIG. 15C is Photo of aPrototype Microchip Developed for Mass Production of Microspheres (SideView).

There are shown in FIG. 16 and FIG. 17 a block diagram for describingthe flow control principle of the flow control unit of the massproduction apparatus in accordance with an embodiment of the presentinvention and a graph illustrating a flow rate control principle of theflow control unit of the mass production apparatus according to anotherembodiment of the present invention, respectively.

FIG. 16 is Block Diagram for describing a Flow Control Principle of theFlow Rate Control Unit of the Mass Production Apparatus. FIG. 17: Graphillustrating the Flow Rate Control Principle of the Flow Rate ControlUnit of the Mass Production Apparatus.

In FIGS. 16 and 17, the pressure P2 of the first material outlet 320 ofthe first material reservoir 300 can be expressed by the followingequation:

P2=P1+ρgh

wherein P1 is the first gas pressure introduced into the first materialreservoir 300, p, the density of the first raw material, g, thegravitational acceleration, and h, the height from the first materialoutlet 320 to the top surface of the first raw material. That is, thepressure P2 of the first material outlet 320 can be represented by thesum of the pressure of the gas layer above the first material reservoir300 or the first gas pressure P1 to be introduced and the hydrostaticpressure (σgh) of the first raw material at the first material outlet320.

In closed systems of incompressible fluids, for example, for liquid suchas water or organic solvents, the flow rate introduced and the flow ratedischarged are known to be the same. The flow rate inside the closedsystem depends on the pressure difference or pressure gradient at theinlet and outlet of the closed system.

In an embodiment of the present invention, since a complete airtightnesscan be maintained at the multichannel microsphere forming unit 100except for at the inlet and the outlet, the multichannel forming unit100 can be considered as a closed system. Further, in an embodiment ofthe present invention, a constant pressure, for example, an atmosphericpressure level, can be maintained at the outlet of the multichannelforming unit 100. Accordingly, if both pressure P2 of the first materialoutlet 320 delivering the raw material to the multichannel microsphereforming unit 100 and pressure of the second material outlet 420 are keptconstant, the flow rate of the fluids running through the multichannelmicrosphere forming unit 100 can be held constant.

During the production of the microspheres using the apparatus accordingto an embodiment of the present invention, the first material inlet 310is to be closed using the first material inlet valve 312, allowing thefirst material reservoir 300 to be in fluid communication only with thefirst flow control line 210 and the first material outlet 320, resultingin the flow rate of the first raw material flowing through the firstmaterial outlet 320 corresponding to that of the first gas introducedinto the first material reservoir 300. In the graph of FIG. 17, thefirst gas flow rate is illustrated as being equal to the flow rate ofthe first raw material discharged to the first material outlet 320.Further, in the graph of FIG. 17, the discharge flow rate of the firstgas and the pressure P1 of the first gas introduced are shown asfluctuations having a relatively small amplitude. The flow rate and thepressure P1 of the first inlet gas will fluctuate at a frequencycorresponding to the cycle of the flow rate control unit 200 as aconsequence of the flow rate control unit 200 may be being, for example,a mechanical pump operating at a constant frequency or cycle, and thefirst gas is compressible.

If the pressure P2 of the first material outlet 320 fluctuates accordingto the pressure P1 of the first gas introduced, it results in both theflow rate discharged through the first material outlet 320 and the flowrate delivered to the multichannel microsphere forming unit 100fluctuating accordingly.

In an embodiment of the present invention, the first gas is introducedinto the gas layer above the first flow reservoir through the first flowcontrol line of the flow rate control unit 200. As a consequence of thegas layer above the first flow reservoir, however, having a volumegreater than the flow rate of the first gas introduced, the pressurefluctuation of the introduced first gas can be leveled off or attenuatedin the entire gas layer above the first flow reservoir, allowing theupper gas layer to press down the surface of the first raw material at auniform pressure without fluctuation, allowing the pressure P2 and flowrate of the first material outlet 320 to be kept constant, i.e., withoutfluctuation.

Further, as the microspheres get formed in the apparatus in accordancewith an embodiment of the present invention, the fluid level h of thefirst raw material stored in the first material reservoir 300 willgradually decrease, resulting in a corresponding decrease in the thehydrostatic pressure ρgh due to the weight of the first raw material,which, in turn, as a consequence of the reduced fluid level h and thehydrostatic pressure ρgh, results in a corresponding increase in thepressure of the introduced first gas which was initially introduced intothe multichannel microsphere forming unit 100 from the flow control unit200 at a constant flow rate.

In order for the microspheres formed in the multichannel microsphereforming unit 100 to have a narrow size distribution or monodispersed,the process parameters of the multichannel microsphere forming unit 100is strictly controlled, in particular, the flow rates therewithin. Ifthe flow rate of the raw materials introduced into the multichannelmicrosphere forming unit 100 is not constant or fluctuates in a shortperiod, i.e., fluctuates rapidly, it results in the microsphere formingconditions varying accordingly and the microspheres formed having abroad size distribution.

For a mass production of microspheres involving a multichannelmicrosphere forming unit 100 incorporating therein, for example, morethan hundred microchannels, a relatively large amount of raw matereials,for example, the first and second raw materials in the presentembodiment, need to be introduced thereinto, as compared tolaboratory-scale production equipment involving a multichannel formingunit 100 incorporating therein only a few microchannels. In addition,the frequency of process interruptions due to a raw material replacementshould be minimized in mass production.

Further, in an embodiment of the present invention, in order for themicrosphere forming process to be maintained for an extended period oftime, a significant amount of the first raw material that have beensterilized is stored in the first material reservoir 300. By keeping theflow rate of the first gas supplied to the first material reservoir 300constant, it is possible to keep constant the flow rate of the first rawmaterial discharged from the first material reservoir 300, that is, thefirst raw material delivered to the multichannel microsphere formingunit 100, using the flow rate control unit 200. In addition, the flowrate can be smoothly maintained, i.e., without fluctuation, by forming agas layer, for example, a gas buffer layer, on the upper portion of thefirst material reservoir 300 and by supplying the first gas to the gaslayer through the flow rate control unit 200 and the first flow controlline 210.

The flow rate control unit 200 and the mass production apparatusformicrospheres according to an embodiment of the present invention aredescribed using the first material reservoir 300 shown in FIGS. 16 and17. The flow control of the second material reservoir 400 may beidential to the one for the first material reservoir 300.

Referring again to FIG. 14, the multichannel forming unit 100 of theapparatus according to an embodiment of the present invention receivesthe first raw material from the first material reservoir 300 and receivethe second raw material from the second material reservoir 400. Themultichannel microsphere forming unit 100 includes a plurality ofmicrochannels through which the first raw material and the second rawmaterial flow respectively. Using the principle as described above inFIGS. 2 through 5, microspheres are formed by the interaction of thefirst raw material and the second raw materials flowing through therespective microchannels and merging at the merging point thereof.

Formed microspheres are transferred to the product reservoir 500 throughthe product outlet 110, the product reservoir 500 including therein asolution that is similar or identical to the first raw material storedin the first material reservoir 300 such as, for example, PVA dissolvedpurified water. The dispersion solution reservoir 700 comprising adispersion solution introduces the dispersion solution to the productreservoir through the dispersion solution inlet valve 712. After thedispersion solution contained in the dispersion solution reservoir 700is sufficiently introduced into the product reservoir 500, thedispersion solution inlet valve 712 mounted in the product reservoir 500is closed. Thereafter, the product inlet valve 512 is opened, connectingthe product inlet 510 of the product reservoir 500 to the product outlet110 of the multichannel microsphere forming unit 100. The microspheresformed in the multichannel microsphere forming unit 100 are introducedinto the dispersion solution stored in the product reservoir 500, thedispersion solution preventing the newly produced microspheres frombeing aggregated. The product inlet valve 512 is to be closed after thecompletion of the microsphere manufacturing process. Thereafter, thedispersion solution containing the produced microspheres are to bedischarged through the outlet 520 to be further processed or treated,such as sieving and filtering, depending on the final productrequirements.

FIG. 18 is Block Diagram illustrating a Channel-to-Channel FluidicConnection Relationship in the Multichannel Microsphere Forming Unit ofthe Mass Production Apparatus according to an exemplary embodiment ofthe present invention.

There is shown in FIG. 18 a multichannel microsphere forming unit 100 ofthe mass production apparatus according to an embodiment of the presentinvention comprising a first inlet manifold 120, a second inlet manifold130, a plurality of first microchannels, a plurality of secondmicrochannels and a plurality of third microchannels.

Detailed specifications of individual microchannels and principles ofmicrosphere formation have been described above with reference to FIGS.3 to 5. For the sake of brevity, repeated descriptions relating theretoare omitted.

In the embodiment, the first inlet manifold 120 for receiving the firstraw material delivered from the first material reservoir 300 is in fluidcommunication with the first material outlet 320 of the first materialreservoir 300. The first inlet manifold 120 is also in fluidcommunication with the plurality of first microchannels 122;122_1-122_nand supplies the received first raw material to each of the firstmicrochannels. The second inlet manifold 130 for receiving the secondmaterial from the second material reservoir 400 is in fluidcommunication with the second material outlet 420 of the second materialreservoir 400. The second inlet manifold 130 is also in fluidcommunication with the plurality of second microchannels 132;132_1-132_nand supplies the received second raw material to each of the secondmicrochannels. The second inlet manifold 130 is not directly connectedto the plurality of the first microchannels 122;122_1-122_n. In thisregard, a plurality of first microchannels 122;122_1-122_n which passunder the second inlet manifold 130 is depicted with dotted lines inFIG. 18.

Respective first microchannel and second microchannel are merged at aplurality of merging points 124;124_1-124_n. A plurality of thirdmicrochannels 126;126_1-126_n are connected to a plurality of mergingpoints 124;124_1-124_n, respectively, the plurality of thirdmicrochannels 126;126_1-126_n extending from the plurality of mergepoints 124;124_1-124_n to the product outlet 110. The first raw materialflowing through the plurality of first microchannels 122;122_1-122_n andthe second raw material flowing through the plurality of secondmicrochannels 132;132_1-132_n are to be merged at the plurality ofmerging points 124;124_1-124_n, resulting in the formation ofmicrospheres thereat. The mixed solution of the first raw material andthe second raw material including therein the formed microspheres flowthrough the plurality of third microchannels 126;126_1-126_n to becollected at the product outlet 110.

For a mass production, the multichannel microsphere forming unit 100 ofthe apparatus according to an embodiment of the present invention mayneed to incorporate therein a large number of microchannels, forexample, over a hundred microchannels. In order for the microspheresformed in each of the microchannels to be of an even sized, that is,microspheres having a narrow size distribution or monodispersed,important parameters in microsphere formation in each microchannel, inparticular, the flow rate of the materials therein and the dimensionthereof, should be strictly controlled.

As described above, the size and the size distribution of themicrospheres formed are mainly determined by few parameters, forexample, the polymer concentration in each of the solutions, the flowrate of the materials and the consistency thereof in the microchannelsand the dimension of the microchannels.

In an embodiment of the present invention, the concentration and theviscosity of the respective raw materials entering each of the dedicatedmicrochannels are substantially the same for the first raw material andthe second raw material because the first and second raw materialsflowing into the plurality of first or second microchannels122;122_1-122_n, 132;132_1-132_n are supplied from the first or secondmaterial reservoirs 300, 400 after being stirred enough therein to beuniformly mixed. Further, in an embodiment of the present invention, themultichannel microsphere forming unit 100 can be formed with highdimensional accuracy on a rigid material such as silicon wafer, glass orPDMS through semiconductor processes. As is known, current semiconductorprocesses are capable of providing ultra-fine microchannel structuresmuch smaller than 50 μm. Since the microchannels of the multichannelmicrosphere forming unit 100 are formed of the same semiconductormaterial using ultra-fine processes, the microchannels therein have theuniform channel dimensions and wall wettability.

Once the diameter of the microspheres desired is set, the first step inoptimizing the mass production of microspheres is to design themultichannel microsphere forming unit 100, based on the resultsdisclosed hereinabove, to be incorporated in the mass productionapparatus, and the first step in designing the multichannel forming unit100 is to fix the material constituting the multichannel microsphereforming unit 100, for this will fix the wall wettability. As the nextstep, the microchannel dimension needed should get fixed, for and themicrochannel dimension is the next easiest parameter to control, and themicrochannel dimension must meet, according to the results obtained anddescribed herein above, one of the following criteria: (1) the crosssectional area of the microchannel should be within 30% of the crosssectional area of the microsphere to be formed; (2) in case that crosssection of the microchannel is a square, i.e., the width and the depthare the same, the width and the depth of the microchannel should bewithin 30% of the diameter of the microsphere to be formed; or (3) incase that the cross sectional of the microchannel is not square, ie., arectangle, at least one of the side thereof should be within within 30%of half of the diameter of the microsphere to be formed.

In order to obtain microspheres with uniform size and narrow sizedistribution, i.e., monodisperse, strict control of the flow rate ineach of the microchannels in the multichannel microsphere forming unit100 should be tightly controlled. If the introduction pressure of thefirst or second raw material is different for each microchannel at thepoint where the first or second raw material is introduced into each ofthe plurality of first or second microchannels 122;122_1-122_n,132;132_1-132_n, it results in the flow rate through the channel varyingfrom microchannel to microchannel. In addition, if the inlet pressurechanges or fluctuates over time, it results in the flow rate in themicrochannels varying at the plurality of merging points124;124_1-124_n, where the microspheres are fomed, leading a wide sizedistribution in the microspheres formed.

In an embodiment of the present invention, the first and second manifold120, 130 has a volume that is considerably larger than the size and theflow rate of the plurality of first and second microchannels122;122_1-122_n, 132;132_1-132_n, respectively. More specifically, in anembodiment of the present invention, the first inlet manifold 120 has avolume sufficiently large enough to provide substantially the same flowrate to the plurality of first microchannels 122;122_1-122_n, and thesecond inlet manifold 130, a volume large enough to providesubstantially the same flow rate to the plurality of secondmicrochannels 132;132_1-132_n.

As can be seen in FIG. 18, the path for the flow of the first rawmaterial at the leftmost microchannel is shorter than the same at therightmost microchannel as the latter path includes an additional pathfor the flow inside the first inlet manifold 120, wherein the leftmostmicrochannel path is from the first path of the first microchannel andthe first path of the third microchannel to the product outlet 110, andthe rightmost channel path is from the nth path of the firstmicrochannel and the nth path of the third microchannel to the productoutlet 110. Unless the first and the second inlet manifold 120, 130 havesufficiently large volume, the pressure gradient along the length of thefirst or the second inlet manifold 120, 130 could occur, resulting theinlet pressure at each microchannel being different from one another.

However, inside pressure of each manifold according to an embodiment ofthe present invention is constant overall. The first or the second inletmanifold 120, 130 has considerably larger volume compared to the sizesand the flow rates of the plurality of first or second microchannels122; 122_1-122_n, 132;132_1-132_n. The pressures within each manifoldcould be considered constant by averaging effect over the entire volumeof the manifold, reasulting in the first and the second inlet manifold120, 130 functioning as a pressurized source for each dedicatedmicrochannels, the plurality of first microchannels 122;122_1-122_n andthe plurality of second microchannels 132;132_1-132_n, respectively.That is, the inlet pressure of the first raw materials from the firstinlet manifold 120 to each of the first microchannels 122;122_1-122_nare substantially the same, and the same applies to the second inletmanifold 130. Such a manifold having considerably larger volume providesa constant pressure of the first or second raw material introduced intothe first or second microchannels 122;122_1-122_n, 132;132_1-132_n,respectively, without fluctuation during the whole process.

Thus, in an embodiment of the present invention, each pressure gradientsalong the plurality of first microchannels 122; 122_1-122_n or theplurality of second microchannels 132;132_1-132_n are substantiallyidentical and are constant during the process, resulting in the flowrates being substantially the same throughout the process.

There are shown in FIGS. 19 to 24 an assembled perspective view of amultichannel microsphere forming unit of the mass production apparatusfor microspheres according to an embodiment of the present invention, anexploded perspective view of the multichannel microsphere forming unitof FIG. 19, an exploded perspective front view of the multichannelmicrosphere forming unit of FIG. 19, an assembled perspective front viewof the multichannel microsphere forming unit of FIG. 19, a bottom viewof the upper case of FIG. 19 and a top view of the lower case of FIG.21, respectively.

FIG. 19 is Assembled perspective view of the Multichannel MicrosphereForming Unit of the Mass Production Apparatus according to an embodimentof the present invention. FIG. 20 is Exploded perspective view of theMultichannel Microsphere Forming Unit of FIG. 19. FIG. 21 is Explodedperspective front view of the Multichannel Microsphere Forming Unitshown in FIG. 19. FIG. 22 is Assembled perspective front view of theMultichannel Microsphere Forming Unit shown in FIG. 19. FIG. 23 isBottom view of the Upper Case shown in FIG. 19. FIG. 24 is Top view ofthe Lower Case shown in FIG. 21.

Referring FIGS. 17 to 22, the multichannel microsphere forming unit 100of the mass production apparatus for microspheres according to anembodiment of the present invention comprises an upper case 1100, alower case 1200, a plurality of O-rings OL1, OL2, an upper multichannelplate 1300, a lower multichannel plate 1400 and product outlets 1500,1510. In assembly, the upper case 1100 and the lower case 1200 arefastened to each other. The plurality of O-rings OL1, OL2, the uppermultichannel plate 1300 and the lower multichannel plate 1400 aredisposed between the upper case 1100 and the lower case 1200. The uppercase 1100 and the lower case 1200 are made of a corrosion-resistantmaterial such as stainless steel or a rigid plastic. Although not shown,the upper case 1100 and the lower case 1200 are fastened to each otherby fastening means such as bolts. However, the present invention is notlimited thereto. In some embodiments, the upper case 1100 and the lowercase 1200 are fastened to each other by tightening means such as a clampor adhesive means such as an adhesive or through welding.

The upper case 1100 and the lower case 1200 have the same shape. In theillustrated embodiment, the upper case 1100 and the lower case 1200 arerectangular plates. However, the present invention is not limitedthereto. The upper case 1100 and the lower case 1200 can be in any shapesuch as discs capable of properly positioning the upper multichannelplate 1300 and the lower multichannel plate 1400 therebetween.

The uppercase 1100 comprises a first inlet pipe 1110, a second inletpipe 1120, a first annular manifold 1130 and a second annular manifold1140. The first inlet pipe 1110 and the second inlet pipe 1120 aredisposed on the upper surface of the upper case 1100. The first annularmanifold 1130 and the second annular manifold 1140 are formed on thelower surface of the upper case 1100.

The first inlet pipe 1110 is in fluid connection with the first materialoutlet 320 of the first material reservoir 300 and the other end is influid connection with the first annular manifold 1130 passing throughthe upper case 1100. The first raw material is supplied from the outsideof the multichannel microsphere forming unit 100 through the first inletpipe 1110 and is delivered to the first annular manifold 1130 at thelower surface of the upper case 1100.

The second inlet pipe 1120 is in fluid connection with the secondmaterial outlet 420 of the second material reservoir 400, and the otherend, in fluid connection with the second annular manifold 1140 passingthrough the upper case 1100. The second raw material is supplied fromthe outside of the multichannel microsphere forming unit 100 through thesecond inlet pipe 1120 and is delivered to the second annular manifold1140 at the lower surface of the upper case 1100.

In one embodiment of the invention shown, the first inlet pipe 1110 andthe second inlet pipe 1120 are branched at the upper portion of theupper case 1100, allowing the first annular manifold 1130 or the secondannular manifold 1140 to be connected with two branched pipes,respectively. However, the present invention is not limited thereto. Inother embodiments, the first annular manifold 1130 or the second annularmanifold 1140 can be connected with a single inlet pipe or branchedinlet pipes having more than three branches.

The first annular manifold 1130 and the second annular manifold 1140 arean annular recess formed in the lower surface of the upper case 1100.The first annular manifold 1130 is disposed radially outward of thesecond annular manifold 1140. In the illustrated embodiment, the radialcross-sections of the first annular manifold 1130 and the second annularmanifold 1140 are substantially the same. However, the present inventionis not limited thereto. For example, the second annular manifold 1140can have a larger radial cross-section than the first annular manifold1130, resulting in the volumes of both manifolds being the same or closeto each other.

The upper case 1100 is disposed on the upper portion of the uppermultichannel plate 1300 and the plurality of O-rings OL1, OL2 aredisposed between the upper case 1100 and the upper multichannel plate1300. The plurality of O-rings OL1, OL2 comprise the first O-rings OL1placed radially adjacent to the first annular manifold 1130 inwardly oroutwardly and the second O-rings OL2 placed radially adjacent to thesecond annular manifold 1140 inwardly or outwardly. The plurality ofO-rings OL1, OL2 prevent the fluids from leaking along the boundarylayers between the upper case 1100 and the upper multichannel plate1300, the fluids containing the first raw material or the second rawmaterial. In the illustrated embodiment of the present invention, twofirst O-rings OL1 and two second O-rings OL2 are arranged. However, thepresent invention is not limited thereto. There may be less or morenumber of first O-rings OL1 and second O-rings OL2 provided a propersealing is ensured.

The lower case 1200 includes a plate seating groove 1210 formed on theupper surface of the lower case 1200 and a product-exhausting hole 1220passing through the lower case 1200 from the center of the lower case1200.

The plate seating groove 1210 is formed on the upper surface of thelower case 1200 and is a recess having a shape corresponding to theouter shape of the upper multichannel plate 1300 and the lowermultichannel plate 1400. The plate seating groove 1210 has a disk-likeshape and includes a case alignment portion 1230 formed on one side ofthe circumferential region. The case alignment portion 1230 has a shapein which a part of the circumference of the circle is cut off. The uppermultichannel plate 1300 and the lower multichannel plate 1400 also havea shape corresponding to the shape of the plate seating groove 1210including the case alignment portion 1230, allowing the uppermultichannel plate 1300 and the lower multichannel plate 1400 to bealigned with each other when placed inside the plate seating groove1210.

The product-exhausting hole 1220 is disposed at the center of the lowercase 1200, for example, at the center of the plate seating groove 1210.The microspheres formed inside the multichannel microsphere forming unitare collected at the product-exhausting hole 1220. Theproduct-exhausting hole 1220 may form a common exhausting path for theplurality of microchannels in the multichannel microsphere forming unit100. The product-exhausting hole 1220 is connected to theproduct-exhausting ports 1550, 1510 attached to the lower surface of thelower case 1200.

The product-exhausting ports 1500, 1510 comprise a coupling body 1500and a product-exhausting pipe 1510. The product-exhausting pipe 1510 isfixed to the coupling body 1500 and the coupling body 1500 is fastenedto the lower surface of the lower case 1200. The product-exhausting pipe1510 is in fluid communication with the product-exhausting hole 1220 ofthe lower case 1200. The product-exhausting pipe 1510 extends to theproduct reservoir 500 and is used for collecting the microspheres formedin the multichannel microsphere forming unit 100 and for transferingthem to the product reservoir 500.

FIG. 25 is Top view of the Upper Multichannel Plate of the MultichannelMicrosphere Forming Unit according to an embodiment of the presentinvention.

Referring FIGS. 19 to 20 and 25, the upper multichannel plate 1300comprises a plurality of first channel connection holes 1310, aplurality of second channel connection holes 1320 and a plate alignmentportion for the upper plate.

The plurality of first channel connection holes 1310 are arranged alonga first circle having a first diameter. The plurality of second channelconnection holes 1320 are arranged along a second circle having a seconddiameter smaller than the same of the first diameter. In an embodimentof the present invention, the plurality of first channel connectionholes 1310 are disposed radially outward of the plurality of secondconnection holes, and the plurality of first channel connection holes1310 and the plurality of second channel connection holes 1320 aredisposed of coaxially.

The outer shape and dimensions of the upper multichannel plate 1300correspond to the outer shape and dimensions of the plate seating groove1210 of the lower case 1200 to be mounted. In an embodiment of thepresent invention, the upper multichannel plate alignment portion 1330of the upper multichannel plate 1200 are fitted to the case alignmentportion 1230 of the lower case 1200.

The upper multichannel plate 1300 is made of a rigid material capable ofbeing formed with a high dimensional accuracy, such as a silicon wafer,a glass wafer, PDMS, or the like. In particular, in one embodiment ofthe present invention, the upper multichannel plate 1300 is made of aglass wafer. In an embodiment of the present invention, the plurality offirst channel connection holes 1310 and the plurality of second channelconnection holes 1320 have a relatively simple structure and are formedon the glass substrate having a higher toughness than a silicon wafer.

FIG. 26 is a top view of the lower multichannel plate of themultichannel microsphere forming unit according to an embodiment of thepresent invention. FIG. 26 is Top view of the Lower Multichannel Plateof the Multichannel Microsphere Forming Unit according to an embodimentof the present invention.

Referring FIGS. 19 to 22 and 26, the lower multichannel plate 1400includes the plurality of first microchannels 1410, the plurality ofsecond microchannels 1420, the plurality of third microchannels 1412 anda center through-hole 1440. Further, the lower multichannel plate 1400is disposed between the upper multichannel plate 1300 and the lower case1200.

The plurality of first microchannels 1410, the plurality of secondmicrochannels 1420, and the plurality of third microchannels 1412 have atrench structure formed on the upper surface of the lower plate.

The plurality of first microchannels 1410 are arranged radially on theupper surface of the upper multichannel plate 1300. Each of the firstmicrochannel 1410 is arranged radially from the center through-hole1440. The plurality of second microchannels 1420 are arranged radiallyin parallel with the first microchannel 1410. The plurality of firstmicrochannels 1410 and the plurality of second microchannels 1420 aremerged at the merging point 1414. The plurality of third microchannels1412 are arranged radially inward of the plurality of firstmicrochannels 1410. One end of the third microchannel 1412 is connectedto the merging point 1414 and the other end thereof is connected to thecenter through-hole 1440.

The outer shape and dimensions of the lower multichannel plate 1400correspond to the outer shape and dimensions of the plate seating groove1210 of the lower case 1200 to be mounted. In an embodiment of thepresent invention, the lower multichannel plate alignment portion of thelower multichannel plate 1400 is fitted to the case alignment portion1230 of the lower case 1200.

The lower multichannel plate 1400 is made of a rigid material capable ofbeing formed with a high dimensional accuracy, such as a silicon wafer,a glass wafer, PDMS, or the like. In particular, in an embodiment of thepresent invention, the lower multichannel plate 1400 is made of acrystalline or amorphous silicon wafer. In an embodiment of the presentinvention, microchannels having a high dimensional accuracy areappropriately formed on a silicon wafer as a consequence thereof havinga high dimensional stability.

There are illustrated in FIGS. 27 to 31 a top view showing the uppermultichannel plate overlapped with the lower multichannel plate of themultichannel microsphere forming unit according to an embodiment of thepresent invention, a top surface translucent diagram showing FIG. 27with the first annular manifold and the second annular manifold of theupper case in hidden lines, a cross-sectional view of the upper case,the upper multichannel plate, and the lower multichannel plate takenalong line X-X′ in FIG. 28, a cross-sectional view of the upper case,the upper multichannel plate and the lower multichannel plate along theline Y-Y′ in FIG. 28 and a cross-sectional view of the upper case, theupper multichannel plate, and the lower multichannel plate along theline Z-Z′ in FIG. 28, respectively.

FIG. 27 is Top view showing the Upper Multichannel Plate overlapped withthe Lower Multichannel Plate of the Multichannel Microsphere FormingUnit according to an embodiment of the present invention.

FIG. 28 is Top surface translucent diagram showing FIG. 27 with theFirst Annular Manifold and the Second Annular Manifold of the Upper Casein hidden lines.

FIG. 29 is Cross-sectional view of the Upper Case, the UpperMultichannel Plate and the Lower Multichannel Plate taken along lineX-X′ in FIG. 28.

FIG. 30 is Cross-sectional view of the Upper Case, the UpperMultichannel Plate and the Lower Multichannel Plate along the line Y-Y′in FIG. 28.

FIG. 31 is Cross-sectional view of the Upper Case, the UpperMultichannel Plate and the Lower Multichannel Plate along the line Z-Z′in FIG. 28.

Referring FIGS. 27 to 31, the upper multichannel plate 1300 and thelower multichannel plate 1400 should be completely in contact andaligned when the multichannel forming portion 100 has been assembled.The open upper portion of the microchannels of the lower multichannelplate 1400 having a trench structure is closed off from the outside bythe lower surface of the upper multichannel plate 1300. Thus, asdescribed above, the microchannels for forming the microspheres arelocated between the upper multichannel plate 1300 and the lowermultichannel plate 1400.

In an embodiment of the present invention, the plurality of firstchannel connection holes 1310 of the upper multichannel plate 1300 aredisposed on the plurality of first microchannels 1410 (see FIG. 29). Inaddition, the plurality of second channel connection holes 1320 of theupper multichannel plate 1400 are disposed on the plurality of secondmicrochannels 1420 (see FIG. 30).

The plurality of first microchannels 1410 are in fluid connection withthe first annular manifold 1130 through the respective firstmicrochannels 1410 and the plurality of second microchannels 1420 are influid connection with the second annular manifold 1140 through therespective second microchannel 1420. The plurality of firstmicrochannels 1410 and the plurality of second microchannels 1420 arerespectively merged at the plurality of merging points 1414. Theplurality of third microchannels 1412 are respectively joined at theplurality of merging points 1414 and extend to the center through-hole1440 (see FIG. 30).

The first annular manifold 1130 and the second annular manifold 1140 areprovided with a significantly larger volume relative to the size andflow rate of the plurality of first microchannels 1410 and the pluralityof second microchannels 1420. The pressure inside the first annularmanifold 1130 and the second annular manifold 1140 are averaged over theentire volume and are constant during the process. Thus, the pressure offluids introduced respectively from the plurality of first channelconnection holes 1310 or the plurality of second channel connectionholes 1320 to the plurality of first microchannels 1410 or the pluralityof second microchannels 1420 are kept uniform over the relevant portionsand during the process.

In addition, in an embodiment of the present invention, the plurality offirst microchannels 1410 and the plurality of second microchannels 1420are arranged radially in a plane, that is point-symmetrically withrespect to the center point of the lower multichannel plate 1400.Accordingly, in an embodiment of the present invention, a plurality offlow paths from the plurality of first channel connection holes 1310 tothe center through-hole 1440 through the plurality of firstmicrochannels 1410 and a plurality of third microchannels 1412 are ofthe same first length L1. In addition, a plurality of flow paths fromthe plurality of second channel connection holes 1320 to the centerthrough-hole 1440 through the plurality of second microchannels 1420 andthe plurality of third microchannels 1412 are of the same second lengthL2. That is, the lengths of the corresponding flow paths formed by themicrochannels are of substantially the same length with respect to eachother. As described above, the first annular manifold 1130 or the secondannular manifold 1140 which are in fluid connection with the pluralityof first microchannel connection holes 1310 or the plurality of secondmicrochannel connection holes 1320 are under a constant pressure withouta pressure gradient along the position and are under an uniform pressureduring the process. In addition, since all of the flow paths formed bymicrochannels are commonly connected to the center through-hole 1440 atwhich one same pressure applies, the pressure differences across thecorresponding flow paths formed by the microchannels are substantiallythe same, that is, the pressure gradient of the flowing fluid in thecorresponding flow path is constant for the corresponding channel. In anembodiment of the present invention, the first micochannel connectionhole, the second microchannel connection hole, the first microchannel,the second microchannel and the third microchannel may each be severaltens or more, for example, 100 or more in numbers. Since the flowconditions in the respective microchannels are substantially the same,the microspheres formed will be of the same size, leading to a narrowsize distribution, i.e., monodispersed.

There is shown in FIG. 32 an exemplary schematic diagram illustrating aprocess for forming of microspheres in the mass production apparatusaccording to an embodiment of the present invention.

FIG. 32 is Exemplary schematic diagram illustrating the Forming ofMicrospheres in the Mass Production Apparatus according to an embodimentof the present invention.

There is shown in FIG. 32 an enlarged view of one microsphere formingpath of FIG. 31 and for simplicity of explanation, the size of each ofthe configurations is exaggeratively depicted.

In an embodiment of the present invention, the first annular manifold1130 is connected to the first microchannel 1410 through a firstmicrochannel connection hole 1310. An aqueous solution comprising thefirst raw material, for example, PVA, a surfactant, dissolved in purewater, is supplied to the first microchannels 1410 through the firstannular manifold 1130. The second annular manifold 1140 is connected tothe second microchannels 1420 through a second channel connection hole1320. An aqueous solution comprising the second raw material, forexample, PCL, a biodegradable polymer, dissolved in an oil, is suppliedto the second microchannels 1420 through the second annular manifold1140. At the merge point 1414, the second raw material having ahydrophobic surface is introduced from the second microchannels 1420into the first raw material having a hydrophilic surface. As the amountof the second raw material introduced at the merging point 1414increases, the flow pressure of the first raw material acting on thesecond introduced raw material correspondingly increases, resulting in,at the merging point 1414, the second raw material seperating from thesecond microchannel 1420 and flow in a droplet form along with the firstraw material having a relatively large flow rate in the thirdmicrochannel 1412. An isotropic external force acts on the droplets ofthe hydrophobic second raw material in the hydrophilic first rawmaterial, making it possible for the droplets of the second raw materialto maintain a spherical shape. The droplets thus formed harden withtime, resulting in the formation of the desired microspheres.

If the respective flow rates in the microchannels are maintainedconstant, the microsphere formation process described above can becontinuously repeated, resulting in a constant production cycle,implicating the amount of the first raw material and the second rawmaterials involved in one production cycle to be identical to those inother production cycles, which, in turn, leading to the formation ofmicrospheres that are similar, chemically and physically ormonodisperse.

One of the most critical parameters to be controlled in the massproduction of monodisperse microspheres is the flow rate of thesolutions/fluids and the proposed arrangement provide a unique solutionfor a precise/tight control thereof, namely: (1) a radially arrangedmultichannel structure in the multichannel microsphere forming unit 100capable of providing the same flow length for each corresponding flowpaths for the plurality of microchannels, i.e., capable of ensuring thesame flow length for each corresponding material flowing in therespective microchannel or the same flow rate for each correspondingmaterial flowing in the respective microchannel; and (2) a flow ratecontrol unit 200 capable of supplying the first raw material and thesecond raw material from the respective reervoirs, i.e., the first rawmaterial reservoir 300 and the second raw material reservoir 400, atconstant flow rates to the multichannel microsphere forming unit 100.

Hereinafter, a mass production apparatus of monodisperse microspheresaccording to other embodiments of the present invention will bedescribed. In other embodiments, substantially the same or similarcomponents as those of the technical constructions included in theabove-described embodiment of the present invention are referred to bythe same reference numeral, and repetitive explanations thereof areomitted. Other embodiments of the present invention are described basedon the differences from the embodiments of the present invention. Inother embodiments, the added or modified technical constructions arereferenced with an appended code ‘a,’ ‘b’ and ‘c’ at the end. FIG. 33 isa block diagram of the mass production apparatus for microspheresaccording to another embodiment of the present invention.

Referring to FIG. 33, the apparatus for mass-production of monodispersemicrospheres according to another embodiment of the present inventionfurther includes a third raw material reservoir 600 to the embodiment ofthe present invention shown in FIG. 14.

The third raw material reservoir 600 comprises an oil phase solutiondissolved therein a third raw material, i.e., an organic solvent and abiodegradable polymer dissolved therein. For example, the third materialmay be polyglycolic acid (PGA) with Mn of about 45,000 or less, i.e.,the biodegradable polymer, dissolved in dichloromethane (solvent,melting point 39.6° C.), i.e, oil solvent, at a concentration of 15 wt%., wherein Mn is the number average molecular weight obtained bydividing the molecular weight of the molecular species comprisingpolymeric compounds with molecular weight distribution by the number ormole fraction.

The second raw material and the third raw material may comprisesubstantially the same or similar organic solvent. Illustratively, thesecond material and the third material may have different concentrationsof the biodegradable polymer dissolved in the same organic solvent, ordifferent types of the biodegradable polymer to be dissolved indifferent organic solvent. However, the present invention is not limitedthereto, and the organic solvent of the second material and the thirdmaterials may be composed of other solvents suitable depending on thekind of the biodegradable polymer to be dissolved therein.

The third raw material is sterilized using an approrpiate sterilizationprocess, for example, passing thereof thorugh a sterilization filtrationfilter. The sterilized third raw material is then introduced into thethird material reservoir 600 through a third material inlet 610. Afterthe sterilized third raw material is sufficiently or completelyintroduced into the third material reservoir 600, the third materialinlet valve 612 installed in the third material inlet is shut off,isolating the third material reservoir 600 from the outside, allowingthe sterilization state to be maintained.

The first raw material stored in the first material reservoir 300, thesecond raw material stored in the second material reservoir 400 and thethird raw material stored in the third material reservoir 600 aretransferred to the multichannel microsphere forming unit 100 a throughthe first material outlet 320, the second material outlet 420 and thethird material outlet 620, respectively.

The flow control unit 200 a is respectively in fluid communication withthe first material reservoir 300 through the first flow control line210, with the second material reservoir 400 through the second flowcontrol line 220 and with the third material reservoir 600 through thethird flow control line 230. The flow control unit 200 a introduces afirst gas having a first raw material flow rate into the first materialreservoir 300, a second gas having a second raw material flow rate intothe second material reservoir 400 and a third gas having a third rawmaterial flow rate into the third material reservoir 600. The first gas,the second gas and the third gas are substantially the same kind of gas,for example, clean air or an inert gas.

FIG. 33 is Block diagram of the Microsphere Mass Production Apparatusaccording to another embodiment of the present invention.

The first raw material stored in the first material reservoir 300 isdelivered to the multichannel microsphere forming unit 100 a in anamount corresponding to the first raw material flow rate of theintroduced first gas. The second raw material stored in the secondmaterial reservoir 400 is delivered to the multichannel microsphereforming unit 100 a in an amount corresponding to the second raw materialflow rate of the introduced second gas. In addition, the third rawmaterial stored in the third material reservoir 600 is delivered to themultichannel microsphere forming unit 100 a in an amount correspondingto the third raw material flow rate.

The multichannel microsphere forming unit 100 a The multichannel formingunit 100 a, receiving the first raw material from the first materialreservoir 300, the second raw material from the second materialreservoir 400 and the third raw material from the third raw materialreservoir, includes a plurality of microchannels through which the firstraw material, the second raw material, and the third raw material flowrespectively. In this embodiment, after the initial microspheres areformed through the interaction of the first and the second raw materialsas described above in FIGS. 4 and 5, the third raw material is thencombined with the microspheres formed, for example, through mixing orencapsulation, to form multi-layered microspheres MS2. This known as a“Double Immersion”.

FIG. 34 is Block diagram illustrating a Channel-to-Channel FluidicConnection Relationship of the Multichannel Microsphere Forming Unit ofthe Mass Production Apparatus according to another embodiment of thepresent invention.

Referring to FIG. 34, the multichannel microsphere forming unit 100 a ofthe mass production apparatus according to another embodiment of thepresent invention comprises a first inlet manifold 120, a second inletmanifold, a third inlet manifold, a plurality of first microchannels, aplurality of second microchannels, a plurality of third microchannels, aplurality of fourth microchannels and a plurality of fifthmicrochannels.

The first inlet manifold 120 is in fluid communication with the firstmaterial outlet 320 of the first material reservoir 300 and receives thefirst raw material delivered from the first material reservoir 300. Thefirst inlet manifold 120 is also in fluid communication with theplurality of first microchannels 122;122_1-122_n and supplies thereceived first raw material to each of the first microchannels.

The second inlet manifold 130 is in fluid communication with the secondmaterial outlet 420 of the second material reservoir 400 and receivesthe second raw material S B delivered from the second material reservoir400. The second inlet manifold 130 is also in fluid communication withthe plurality of second microchannels 132;132_1-132_n and supplies thereceived second raw material to each of the second microchannels. Thesecond inlet manifold 130 is not directly connected to the plurality offirst microchannels 122;122_1-122_n. In FIG. 34, the plurality of firstmicrochannels 122;122_1-122_n which pass under the second inlet manifold130 are depicted with dotted lines in FIG. 34. The third inlet manifold140 is in fluid communication with the third material outlet of thethird material reservoir 600 and receives the third raw material S Cdelivered from the third material reservoir 600. The third inletmanifold 140 is also in fluid communication with the plurality of fourthmicrochannels 142;142_1-142_n and supplies the received third rawmaterial to each of the fourth microchannels. The third inlet manifold140 is not directly connected to the plurality of first and secondmicrochannels 122;122_1-122_n 132;132_1-132_n. In FIG. 34, the pluralityof first and second microchannels 122;122_1-122_n 132;132_1-132_n whichpass under the third inlet manifold 140 are depicted with dotted linesin FIG. 34.

Each of the first microchannels 122;122_1-122_n and each of the secondmicrochannels 132;132_1-132_n join each other at the plurality of firstmerging points 124;124_1-124_n. The plurality of third microchannels126;126_1-126_n are joined to the plurality of first merging points124;124_1-124_n, respectively. In addition, the plurality of thirdmicrochannels 126;126_1-126_n extend from the plurality of first mergingpoints 124;124_1-124_n to the plurality of second merging points128;128_1-128_n. The plurality of third microchannels 126;126_1-126_nand the plurality of fourth microchannels 142;142_1-142_n merge witheach other at the plurality of second merging points 128;128_1-128_n.The core microspheres MS1 are formed at the the plurality of the firstmerging points as a consequence of the first raw material flowing in thefirst microchannels merging with the second raw material flowing in theplurality of the second microchannels and flow through the plurality ofthird microchannels. The multi-layered microspheres MS2 are formed atthe plurality of second merging points 128;128_1-128_n as a consequenceof the core microspheres flowing in the pluraity of third microchannelsmerging with the third raw material flowing in the plurality of fourthmicrochannels at the plurality of second merging points 128;128_1-128_n.The multi-layered microspheres thus formed, i.e., MS2, then flow throughthe plurality of fifth microchannels to be collected at the productoutlet 110.

In another embodiment of the present invention, the first inlet manifold120, the second inlet manifold 130, and the third inlet manifold 140 areprovided with a relatively large volume compared to the size of and theflow rate in the plurality of first microchannels n122;122_1-122_n, theplurality of second microchannels 132;132_1-132_n and the plurality offourth microchannels 142;142_1-142_n. More specifically, in anotherembodiment of the present invention, each of the first inlet manifold120, the second inlet manifold 130 and the third inlet manifold 140specifically has a volume sufficiently large enough to providesubstantially the same flow rate to the plurality of first microchannels122;122_1-122_n, the plurality of second microchannels 132;132_1-132_nand the plurality of fourth microchannels 142;142_1-142_n, respectively.

Thus, in another embodiment of the present invention, the plurality offirst microchannels 122;122_1-122_n, the plurality of secondmicrochannels 132;132_1-132_n, or the plurality of fourth microchannels142;142_1-142_n have substantially uniform and constant pressure andflow rate in the respective microchannels.

There are shown in FIG. 35 to FIG. 38 an assembled perspective view ofthe multichannel microsphere forming unit according to anotherembodiment of the present invention, an exploded perspective view of themultichannel microsphere forming unit of FIG. 35, an explodedperspective front view of the multichannel microsphere forming unit ofFIG. 35 and a bottom view of the upper case of FIG. 35, respectively.

Referring FIGS. 35 to 38, the multichannel microsphere forming unit 1000a of the mass production apparatus according to another embodiment ofthe present invention comprises an upper case 1100 a, a lower case 1200,a plurality of O-rings OL1, OL2, OL3, an upper multichannel plate 1300a, a lower multichannel plate 1400 a, and a product exhausting port1500.

In another embodiment of the present invention, the upper case 1100 acomprises a first inlet pipe 1110, a second inlet pipe 1120, a thirdinlet pipe 1150, a first annular manifold 1130, a second annularmanifold 1140, and a third annular manifold 1160. The first inlet pipe1110, the second inlet pipe 1120, and the third inlet pipe 1150 aredisposed on the upper surface of the upper case 1100 a. The firstannular manifold 1130, the second annular manifold 1140, and the thirdannular manifold 1160 are formed on the lower surface of the upper case1100 a.

The first inlet pipe 1110 is in fluid connection with the first materialoutlet 320 of the first material reservoir 300 and the other end is influid connection with the first annular manifold 1130 passing throughthe upper case 1100 a. The first raw material is supplied from theoutside of the multichannel forming unit 100 through the first inletpipe 1110 and is delivered to the first annular manifold 1130 at thelower surface of the upper case 1100 a.

The second inlet pipe 1120 is in fluid connection with the secondmaterial outlet 420 of the second material reservoir 400, and the otherend is in fluid connection with the second annular manifold 1140 passingthrough the upper case 1100 a. The second raw material is supplied fromthe outside of the multichannel forming unit through the second inletpipe 1120 and is delivered to the second annular manifold 1140 at thelower surface of the upper case 1100 a.

The third inlet pipe 1150 is in fluid connection with the third materialoutlet of the third material reservoir 600, and the other end is influid connection with the third annular manifold 1160 passing throughthe upper case 1100 a. The third raw material is supplied from theoutside of the multichannel microsphere forming unit 1000 a through thethird inlet pipe 1150 and is delivered to the third annular manifold1160 at the lower surface of the upper case 1100 a.

In FIG. 38, it is depicted that although the first inlet pipe 1110, thesecond inlet pipe 1120 and the third inlet pipe 1150 are branched at anupper portion of the upper case 1100 a so that two lines arerespectively connected to the first annular manifold 1130, the secondannular manifold 1140 or the third annular manifold 1160, the presentinvention is not limited thereto. In other embodiments, the first inletpipe 1110, the second inlet pipe 1120, and the third inlet pipe 1150 maynot be branched or branched into three or more lines to form aconnection to the first annular manifold 1130, the second annularmanifold 1140 or the third annular manifold 1160.

The first annular manifold 1130, the second annular manifold 1140 andthe third annular manifold 1160 are annular recesses formed on thebottom surface of the upper case 1100 a. The first annular manifold 1130is disposed radially outward of the second annular manifold 1140 and thesecond annular manifold 1140 is disposed radially outward of the thirdannular manifold 1160. In the illustrated embodiment, the radialcross-sections of the first annular manifold 1130, the second annularmanifold 1140, and the third annular manifold 1160 are shown as beingsubstantially the same. However, the present invention is not limitedthereto. For example, the size of the radial cross-sections or the widthof the channels may be different with each other so that the firstannular manifold 1130, the second annular manifold 1140, and the thirdannular manifold 1160 have the same or comparably close sized volume.

The upper case 1100 a is disposed on the upper portion of the uppermultichannel plate 1300 a, and a plurality of O-rings OL1, OL2, OL3 aredisposed between the upper case 1100 a and the upper multichannel plate1300 a. The plurality of O-rings OL1, OL2, OL3 comprise a first O-ringOL1 radially disposed inwardly or outwardly adjacent to the firstannular manifold 1130, a second O-ring OL2 radially disposed inwardly oroutwardly adjacent to the second annular manifold 1140 and a thirdO-ring OL3 radially disposed inwardly or outwardly adjacent to the thirdannular manifold 1160. The plurality of O-rings OL1, OL2, OL3 preventthe leakage of fluids including the first raw material, the second rawmaterial or the third raw material inside the first annular manifold1130, the second annular manifold 1140 or the third annular manifold1160 along the interface between the upper case 1100 a and the uppermultichannel plate 1300 a. In the illustrated embodiment of the presentinvention, a pair of first O-rings OL1, a pair of second O-rings OL2,and a third O-ring OL3 are depicted. However, the present invention isnot limited thereto, for the number of O-rings OL1, OL2, OL3 can beincreased or decreased as long as an appropriate sealing can be assuredtherewith.

FIG. 39 is a top view of an upper multichannel plate of the multichannelmicrosphere forming unit according to another embodiment of the presentinvention.

Referring to FIG. 39, the upper multichannel plate 1300 a includes aplurality of first channel connection holes 1310, a plurality of secondchannel connection holes 1320, a plurality of third channel connectionholes 1350 and a plate alignment portion 1330 for the upper plate.

The plurality of first channel connection holes 1310 are disposed alonga first circle having a first diameter. The plurality of second channelconnection holes 1320 are disposed along a second circle having a seconddiameter smaller than the first diameter. The plurality of third channelconnection holes 1350 are disposed along a third circle having a thirddiameter smaller than the second diameter. In an embodiment of thepresent invention, the plurality of first channel connection holes 1310are disposed radially outward of the plurality of second channelconnection holes 1320, and the plurality of second channel connectionholes 1320 are disposed radially outward of the third channel connectionholes 1350. Further, the plurality of first channel connection holes1310, the plurality of second channel connection holes 1320 and theplurality of third channel connection holes 1350 are disposed ofcoaxially.

The outer shape and size of the upper multichannel plate 1300 acorrespond to the outer shape and size of the plate seating groove 1210of the lower case to be mounted. In an embodiment of the presentinvention, the plate alignment portion of the upper multichannel plateare fitted to the case alignment portion of the lower case.

There are shown in FIGS. 40 and 41 a top view showing the uppermultichannel plate overlapped with the lower multichannel plate of themultichannel microsphere forming unit 1000 a according to an embodimentof the present invention and a top surface translucent diagram showingFIG. 40 with the first annular manifold, the second annular manifold andthe third annular manifold of the upper case in hidden lines,respectively.

Referring to FIGS. 40 and 41, the lower multichannel plate 1400 acomprises a plurality of first microchannels 1410, a plurality of secondmicrochannels 1420, a plurality of third microchannels 1412, a pluralityof fourth microchannels 1450, a plurality of fifth microchannels 1416and a center through-hole 1440. The lower multichannel plate 1400 a isdisposed between the upper multichannel plate 1300 a and the lower case1200.

The plurality of first microchannels 1410, the plurality of secondmicrochannels 1420, the plurality of third microchannels 1412, theplurality of fourth microchannels 1450 and the plurality of fifthmicrochannels 1416 have a trench structure formed on the upper surfaceof the lower multichannel plate 1400 a.

The plurality of first microchannels 1410 are radially disposed on theupper surface of the upper multichannel plate 1300 a. Each of the firstmicrochannels 1410 is disposed radially from the center through-hole1440. The plurality of second microchannels 1420 are radially disposedin parallel with the first microchannels 1410. The plurality of firstmicrochannels 1410 and the plurality of second microchannels 1420 mergewith one another at the first merging points 1414, respectively. Theplurality of third microchannels 1412 are radially disposed inward ofthe plurality of first microchannels 1410. One end of each of the thirdmicrochannels 1412 is connected to the first merging point 1414, and theother end thereof is connected to the second merging point 1418. Theplurality of third microchannels 1412 and the plurality of fourthmicrochannels 1450 are merged at the second merging point 1418. Theplurality of fifth microchannels 1416 are radially disposed inward ofthe plurality of third microchannels 1412. One end of the plurality offifth microchannels 1416 is connected to the second merging point 1418and the other end, to the center through-hole 1440.

The outer shape and size of the lower multichannel plate 1400 acorrespond to the outer shape and size of the plate seating groove 1210of the lower case 1200 to be mounted. In an embodiment of the presentinvention, the plate alignment portion of the lower multichannel plate1440 a is fitted to the case alignment portion 1230 of the lower case1200.

The plurality of first channel connection holes 1310 of the uppermultichannel plate 1300 a are disposed on the plurality of firstmicrochannels 1410. In addition, the plurality of second channelconnection holes 1320 of the upper multichannel plate 1300 a aredisposed on the plurality of second microchannels 1420 and the pluralityof third channel connection holes 1350 of the upper multichannel plate1300 a are disposed on the plurality of fourth microchannels142;142_1-142_n.

The plurality of first microchannels 1410 are respectively in fluidconnection with the first annular manifold 1130 through the plurality offirst channel connection holes 1310, the plurality of secondmicrochannels 1420, respectively in fluid connection with the secondannular manifold 1140 through the plurality of second channel connectionholes 1320, and the plurality of fourth microchannels 1450, respectivelyin fluid connection with the third annular manifold 1160 through thefourth channel connection holes 1350.

FIG. 42 is an exemplary schematic view showing a process for the formingof the multi-layered microspheres MS2 in the mass production apparatusaccording to another embodiment of the present invention.

In FIG. 42, the microsphere forming path is shown in an enlarged form,each configurations being exaggerated in size, enlarged or reduced, forconvenience of explanation.

In another embodiment of the present invention, the first annularmanifold 1130 is connected to the first microchannel through the firstchannel connection hole 1310. The first annular manifold 1130 suppliesthe first raw material, for example, water-phase solution in which PVA,a surfactant, being dissolved in pure water, to the first microchannel1410. The second annular manifold 1140 is connected to the secondmicrochannel 1420 through the second channel connection hole 1320. Thesecond annular manifold 1140 contains the second raw material, forexample, PCL-dissolved oil-phase solution, wherein PCL is abiodegradable polymer. At the first merging point 1414, the second rawmaterial having a hydrophobic surface is introduced from the secondmicrochannel 1420 into the first raw material having a hydrophillicsurface, resulting in a formation of droplets comprising the second rawmaterial, flowing into the third microchannel 1412. As the amount of thesecond raw material introduced at the first merging point 1414increases, the flow pressure of the first raw material acting on thesecond introduced raw material correspondingly increases, resulting in,at the first merging point 1414, the second raw material seperating fromthe second microchannel 1420 and flow in a droplet form along with thefirst raw material having a relatively large flow rate in the thirdmicrochannel 1412. An isotropic external force acts on the droplets ofthe hydrophobic seond raw material in the hydrophillic first rawmaterial, making it possible for the droplets of the second raw materialto maintain a spherical shape. The droplets thus formed harden with timeas they flow in the third microchannels, resulting in the formation ofthe desired microspheres, ie. the core microspheres, i.e., MS1.

The third annular manifold 1160 is connected to the fourth microchannel1450 through the third microchannel connection hole 1350. The thirdannular manifold 1160 may contain a third raw material, for example, anoily solution in which PGA, a biodegradable material, is dissolved in anoil. At the second merging point, the third raw material having ahydrophobic surface is introduced from the fourth microchannel into thefirst raw material having a hydrophilic surface, including therein thecore microspheres, MS1, flowing in the third microchannels 1412. Whenthe core microspheres, i.e., MS1, come in contact with the introducedthird raw material at the second merging point 1418, the hydrophobicthird raw material wets the core microspheres MS1 having a hydrophobicsurface. As the flow pressure acting on the core microspheres MS1 andthe introduced third raw material increases, the core microspheres MS1and the introduced third raw material will be separated from the otherthird raw material in the fourth microchannel 1450. An isotropicexternal force acting on the hydrophobic third raw material in thehydrophilic first raw material spread the third raw material evenlyaround the core microspheres MS1, resulting in the formation ofmulti-layered microspheres MS2 and the multi-layered MS2 thus formedflow in the fifth microchannels 1416.

There are illustarated in FIGS. 43(a) to 43(c) the forming of amulti-layered microsphere MS2 as a consequence of MS1 coming in contactwith the third raw material at the second merging point in accordancewith another embodiment of the present invention and in FIGS. 44(a) to44(c), the forming of a multi-layered microsphere as a consequence ofthe core microsphere coming in contact with the third raw materialintroduced in a relatively large quantity at a second merging point ofthe device according to yet another embodiment of the present invention.

Referring to FIGS. 43(a) and 44(a), at the second merging point 1418where the core microsphere MS1 and the introduced third raw materialcome into contact, the extent to which the third raw material isintroduced may be different. In FIG. 43(a), the third raw materialintroduced is shown to be being relatively small, and in FIG. 44(a), theopposite, i.e., relatively larger amount than shown in FIG. 43(a).

The thickness of the surrounding layer of the third raw materialconstituting the multi-layered microspheres MS2 depends on the amount ofthe third raw material introduced and then separated at the secoondmerging point.

As shown in FIGS. 43(a) to 43(c), when the core microspheres MS1 come incontact with the introduced third raw material at the second mergingpoint 1418, the introduced third raw material (FIG. 43(b)) get attacheditself to the hydrophobic core microsphere MS1 even if the amount of thethird raw material introduced is relatively small (FIG. 43(a)), and MS1with the third raw material attached thereto separate from the secondmerging point 1418 to form the multi-layered microspheres MS2 (FIG.43(c)) when the flow pressures of the first raw material acting thereonbecome sufficiently large.

In contrast, as shown in FIGS. 44(a) to 44(c), when the third rawmaterial in a relatively larger amount than the one shown in FIG. 43(a)comes in contact with the core microspheres MS1, the core microspheresMS1 remains attached to the third raw material for a relatively shorterperiod of time compared to the case shown in FIG. 43(b) before beingseparated from the second merging point 1418 to form the multi-layeredmicrosphere MS2 (FIG. 44(c)) due to the flow pressure of the first rawmaterial.

In the illustrated examples, the amount or thickness of the third rawmaterial forming on the core microsphere MS1 to form the multi-layeredmicrospheres MS2 depends on the amount of the third raw materialintroduced to and separated from the second merging point 1418. Further,it also depends on the flow resistance acting on the third raw materialand the core microsphere MS1 at the moment of separation.

Thus, according to another embodiment of the present invention, if thedimensions and the flow rates of the channels can be kept constant, thesize, the distribution and the layer thickness of the multi-layeredmicrospheres MS2 can be also made constant, independent of the amount ofthe third raw material.

As described above, as a consequence of the present invention capable ofproviding, controlling and maintaining a constant flow rate within theplurality of microschannels, it is possible to generate themulti-layered microspheres using therewith of a desired shape, size anddispersion.

FIG. 45 is a block diagram of the mass production apparatus formicrospheres according to another embodiment of the present invention.

There is shown in FIG. 45, a mass production apparatuss according toanother embodiment of the present invention incorporating therein avision monitoring unit 800 as compared with the embodiment of thepresent invention shown in FIG. 14.

The vision monitoring unit 800 for is disposed on at least one side ofthe multichannel microsphere forming unit 100 b. The vision monitoringunit 800 comprises a camera, for example, a CCD camera, which observesthe state of microsphere formation in the multichannel microsphereforming unit 100 b in real time. The vision monitoring unit 800transmits the visual information/data to an operator viewing unit (notshown). The operator viewing unit may further comprise, for example, adisplay for displaying the photographed images. The operator may analyzethe image/data obtained by and through the vision monitoring unit 800and control the operation of the entire apparatus accordingly basedthereon.

However, the present invention is not limited thereto. In someembodiments, the visual information obtained by the vision monitoringunit 800 may be analyzed in real time using an automatic analyzingapparatus, and the apparatus may be automatically controlled or an alarmmay be sent to the operator according to the analyzed results.

There are shown in FIGS. 46 to 50 an assembled perspective view of themultichannel microsphere forming unit of the mass production apparatusaccording to another embodiment of the present invention, an explodedperspective view of the multichannel microsphere forming unit of FIG.46, an exploded perspective front view of the multichannel microsphereforming unit of FIG. 46, an assembled perspective front view of themultichannel microsphere forming unit of FIG. 46 and a bottom view ofthe upper case of FIG. 46, respectively.

Referring FIGS. 46 to 48, the multichannel forming unit 1000 b of themass production apparatus according to another embodiment of the presentinvention comprises an upper case 1100 b, a lower case 1200, a pluralityof O-rings OL1, OL2, an upper multichannel plate 1300 b, a lowermultichannel plate 1400, a product exhausting port 1500 and a visionmonitoring unit 800.

In another embodiment of the present invention, the upper case 1100 bcomprises a first inlet pipe 1110, a second inlet pipe 1120, a firstannular manifold 1130, a second annular manifold 1140 and a monitoringopening 1170. The monitoring opening 1170 is disposed at the center ofthe upper case 1100 b. The monitoring opening 1170 may also be formedthrough the upper case 1100 b.

The monitoring opening 1170 is of sufficient size to observe themicrochannels in the central region of the lower multichannel plate 1400a, the diameter thereof being larger than the size of theproduct-exhausting hole 1220.

In another embodiment of the present invention, the upper multichannelplate 1300 b is made of a transparent or translucent material. It ismade of a material which is rigid and has a high dimensional accuracy,such as a silicon wafer, a glass wafer, a PDMS, or the like. Inparticular, in another embodiment of the present invention, the uppermultichannel plate 1300 b is made of a glass wafer. The plurality offirst channel connection holes 1310 and the plurality of second channelconnection holes 1320 are provided with a relatively simple structure sothat they can be suitably formed of the glass having a higher toughnessthan a silicon wafer, allowing the observation of the microchannelsformed in the lower multichannel plate 1400 through the monitoringopening 1170 and the upper multichannel plate 1300 b possible.

The vision monitoring unit 800 comprise a camera body 810, a signalcable 820 and a viewing lens 830, the camera body 810 being attached tothe monitoring opening 1170 on the upper surface of the upper case 1100a, wherein the camera body 710 is a camera, for example, a CCD cameraand is used for observing the forming state of microspheres in realtime.

The signal cable 820 is a signal transmission line for transmitting animage photographed by the camera body 810 to an external device. In someembodiments, the vision monitoring unit 800 may comprise a wirelesscommunication unit (not shown) installed in the camera body 810, thewireless communication unit transmitting the visual information to anexternal monitoring device.

The viewing lens 830 is disposed below the camera body 810 wherein theviewing lens 830 is directed through the monitoring opening 1170 to themicrochannels formed on the upper multichannel plate 1300 b and thelower multichannel plate 1400.

Although not shown, the vision monitoring unit 800 may further compriseillumination unit. The illumination unit may be disposed adjacent to theviewing lens 830 and may provide reflected light that is reflected bythe microchannels and is incident on the viewing lens 830.

FIG. 51 is a photographed image of the microchannels around theproduct-exhausting hole and an enlarged exemplary partial view of thesechannels.

Referring to FIG. 51, the vision monitoring unit 800 takes photographsof the microchannels disposed around the product exhausting port 1220.The shape, size, and size distribution of the microspheres formed ineach microchannel can be identified through photographed images. In FIG.51, some microchannels are shown enlarged. In this exemplary enlargementview, one microchannel is shown to be blocked by an abnormally largemicrosphere. If such microsphere clogs one channel, the total flowthrough that microchannel is expected to decrease. As the flow rate ofthe microchannel is reduced, the microspheres formed therein will have adifferent size than the target size, detrimentally affecting themicrosphere size distribution in the overall product.

As the number of microchannels forming the oversized or undersizedmicrospheres outside the target size increases, the size distribution ofthe microspheres in the final product will increase. This will not onlydegrade the quality of the entire product but will also increase theprocessing load in additional sieving.

According to another embodiment of the present invention, it is possibleto monitor the microchannels as to whether or not the microspheresformed are abnormal in size. If the problematic microchannels exceed acertain critical ratio, the operator may perform actions such as waferreplacement, cleaning of the wafer or inspection of other related parts.Alternatively, the valve may be closed to block the flow atmicrochannels causing the problem. This is described below in detail asanother embodiment of the present invention.

FIG. 52 is a block diagram of the mass production apparatus includingtherein a channel-specific valving function according to still yetanother embodiment of the present invention.

Referring to FIG. 52, the apparatus according to another embodiment ofthe present invention further comprises a valving portion 900 ascompared to the embodiment shown in FIG. 45.

The valving portion 900 is connected to the first material outlet 320 ofthe first material reservoir 300 and the second material outlet 420 ofthe second material reservoir 400, respectively. The valving portion 900receives the first raw material from the first material reservoir 300and branch the supplied first raw material to the multichannelmicrosphere forming unit 100 c through the plurality of first materialtransfer lines 910, 912. In addition, the valving portion 900 receivethe second raw material from the second material reservoir 400 andbranch the supplied second raw material to multichannel microsphereforming unit 100 c through the plurality of second material transferlines 920, 922.

The multichannel microsphere forming unit 100 c comprises themicrochannels connected to the respective transmission lines. If one ofthe transmission lines is closed or opened in the valving portion 900,the flow of the material to the microchannel connected thereto is alsoclosed or opened.

That is, when it is determined that the microspheres formed in thespecific microchannels are not of the deired quality according to themonitoring result of the vision monitoring unit 800, the supplies to thecorresponding microchannels may be cut off using the valving portion 900by the operator or the automatic analyzing apparatus, stopping theformation of the problematic microspheres in the related microchannels.FIG. 53 is an exemplarily block diagram showing the channel-to-channelfluidic connection relationship of the valving portion and themultichannel microsphere forming unit in the embodiment shown in FIG.52.

Referring to FIG. 53, the valving portion 900 comprises a plurality offirst material transfer lines 910, 912, a branching unit 902 forbranching the supplied first raw materials and transferring them to theplurality of first material transfer lines 910, 912, and a plurality offirst material valves 930, 932 connected in series to each of theplurality of first material transfer lines 910, 912.

In addition, the valving portion 900 further comprise a plurality ofsecond material transfer lines 920, 922, a branch unit for branching thesupplied second raw materials and transferring them to the plurality ofsecond material transfer lines 920, 922 and a plurality of secondmaterial valves 940, 942 connected in series to each of the plurality ofsecond material transfer lines 920, 922.

In the illustrated embodiment, the valving portion 900 is illustrated asbranching one feed line to two feed lines. However, the number ofmaterial transfer lines to be branched is only exemplary. In someembodiments, the valving portion 900 may branch a single feed line tothree or more delivery lines.

The multichannel microsphere forming unit 100 c may comprise a pluralityof first inlet manifold 120C_1, 120C_2 and a plurality of second inletmanifold 130C_1, 130C_2. In the illustrated embodiment, the plurality offirst inlet manifold 120C_1, 120C_2 and the plurality of second inletmanifold 130C_1, 130C_2 are shown as being two, respectively. However,the present invention is not limited thereto. The number of manifoldsconstituting the first inlet manifold and the second inlet manifold maybe varied corresponding to the number of the material transfer linesbranched and connected at the valving portion 900.

One of the plurality of first inlet manifolds 120C_1 is in fluidcommunication with one of the first transfer lines 910 and others of theplurality of first inlet manifolds 120C_2 are in fluid communicationwith the respective others of the first material transfer lines 912.

One of the plurality of first inlet manifolds 120C_1 is connected to an/2 number of the first microchannels 122C_1-122C_n/2 and the other oneof the plurality of first inlet manifolds 120C_2 is also connected to an/2 number of the respective first microchannels 122C_n/2+1-122C_n.

Similarly, one of the plurality of second inlet manifolds 130C_1 is influid connection with a second material transfer line 920 and the othersof the plurality of second inlet manifolds 130C_2 are in fluidconnection with the respective others of the second material transferline 922.

One of the plurality of second inlet manifolds 130C_1 is connected to an/2 number of second microchannels 132C_1-132C_n/2, and the other one ofthe plurality of second inlet manifolds 130C_2 is similarly connected toa n/2 number of other respective second microchannels 132C_n/2+1-132C_n.

Each first microchannel and each second microchannel are merged at aplurality of merging points 124C;124C_1-124C_n. The plurality of thirdmicrochannels 126C;126C_1-126C_n are connected to the plurality ofmerging points 124C;124C_1-124C_n, respectively. The plurality of thirdmicrochannels 126C;126C_1-126C_n extend from the plurality of mergingpoints 124C;124C_1-124C_n to the product outlet 110.

There is shown in FIG. 54 a bottom view of an upper case forimplementing a multichannel microsphere forming unit according to theembodiment shown in FIG. 53.

Referring to FIG. 54, the upper case 1100C comprises a plurality offirst inlet manifolds 1130C_1, 1130C_2 and a plurality of second inletmanifolds 1140C_1, 1140C_2 formed on a lower surface thereof.

The plurality of first inlet manifolds 1130C_1, 1130C_2 are connected tothe plurality of first inlet pipes 1110C_1, 1110C_2, respectively. Theplurality of second inlet manifolds 1140C_1, 1140C_2 are connected tothe plurality of second inlet pipes 1120C_1, 1120C_2, respectively. Theplurality of first inlet manifolds 1130C_1, 1130C_2 and the plurality ofsecond inlet manifolds 1140C_1, 1140C_2 are of semicircular trenchstructure formed on the lower surface of the upper case 1100C.

FIG. 55 is a block diagram of the mass production apparatusincorporating therein a buff tank according to still another embodimentof the present invention.

Referring to FIG. 55, the apparatus according to another embodiment ofthe present invention further includes a first buffer tank 8000 and asecond buffer tank 9000 as compared to the embodiment shown in FIG. 14.

The first buffer tank 8000 is disposed between the first materialreservoir 300 and the multichannel microsphere forming unit 100. Thefirst buffer tank 8000 is connected to the first material outlet 320 ofthe first material reservoir 300 and receives the first raw materialfrom the first material reservoir 300.

Likewise, the second buffer tank 9000 is disposed between the secondmaterial reservoir 400 and the multichannel microsphere forming unit100. The second buffer tank 9000 is connected to the second materialoutlet 420 of the second material reservoir 400 and the second rawmaterial is supplied thereto from the second material reservoir 400.

As described above, the flow pressures of the first raw material and thesecond raw material transferred through the first material exhaustingport 320 and the second material exhausting port 420 are associated withthe level of the first raw material or the second raw material in thefirst material reservoir 300 or the second material reservoir 400. Thefirst buffer tank 8000 and the second buffer tank 9000 have aconsiderably smaller volume than the first and second materialreservoirs 300, 400. The first buffer tank 8000 and the second buffertank 9000 are installed to relieve the pressure components in accordancewith the liquid level of the first raw material and the second rawmaterial, making it possible to stabilize the flow rate supplied to themultichannel microsphere forming unit 100, i.e., to maintain thepressure thereto constant.

The first buffer tank 8000 further comprises a first material shut-offvalve 8100 and the second buffer tank 9000 further comprises a secondmaterial shut-off valve 9100.

The first material shut-off valve 8100 or the second material shut-offvalve 9100 shut off the first buffer tank 8000 or the second buffer tank9000 from the first material reservoir 300 or the second materialreservoir 400 when the first material reservoir 300 or the secondmaterial reservoir 400 need to be replaced or to be charged with rawmaterials, making it possible to maintain the continuous formation ofmicrospheres until the raw materials in the first buffer tank 8000 orthe second buffer tank 9000 are exhausted without supplying of the rawmaterials from the first material reservoir 300 or the second materialreservoir 400. The presense of the shut-off valves facilitates thecontinuous forming of the mcirospheres during, for example, during theshut-off, the need for the first material reservoir 300 or the secondmaterial reservoir 400 to be replaced or filled.

In addition, the presence of the first buffer tank 8000 and the secondbuffer tank 9000 prevent the loss in cost due to the disposal of the rawmaterials in case of a failure or a maintenance which may occur duringthe manufacturing process.

Medical Products Developed Using the Proposed Principles and the MassProduction Apparatus Developed Based Thereon

The inventors have developed the basic principles for optimizing themass production of monodisperse biodegradable microspheres and have comeup with an apparatus dedicated therefor based on the basic principlesdeveloped. The apparatus developed along with the basic principles canbe easily be used for the mass production of a variety of medicalproducts based on biodegradable polymer microspheres with the desiredphysiological functions/efficacies by mere addition and/or proper mixingof a substance having a desired physiological function or apharmacological function to a biodegradable polymer phase solutionformed by dissolving a biodegradable polymer in an organic solvent andforcing the flow of the biodegradable polymer solution incorporatingtherein the substance to come into a physical contact with the flow of asolution which is immiscible thereto, resulting in the formation ofmicrospheres having the desired properties.

The inventors have applied the developed principles and the apparatus tofabricate three exemplary medical products, namely, medical filler,heartworm preventive and hair loss preventive, all of which areincorporate therein the biodegradable polymer-based microspheres formedusing therewith The medical products developed all have two commonfeatures, namely, being able to be injected into the body and themedical products, or to be more specific, the biodegradable polymertherein degrades over time, i.e., during its biodegradation period.

Further, excluding the medical filler, the heartworm preventive and thehair loss preventive, as a consequence of the substance having thedesired physiological function or a pharmacological function beingreleased into the body as the biodegradable polymer degrades during itsdegradation period, qualifying the products to be polymeric drugdelivery systems (PDDS). In other word, the medical products developedbased on biodegradable polymer-based microspheres serve as injectableimplants in the body and may also function as polymeric drug deliverysystems (PDDS) exposing the physiological or pharmacological functionthroughout the biodegradation period. To summarize, in order for medicalproducts to be qualified as PDDS, they must fulfill the followingrequirements:

-   -   (1) They are injectable;    -   (2) They have an associated degradation period; and    -   (3) They have an associated drug delivery rate.

The biodegradation period is supposed to depend mainly upon the type andquantity of the biodegradable polymer being used in forming themicrospheres. Thus, if the type is defined, for example, as PCL, thebiodegradation period will be determined by the quantity of thebiodegradable polymer used. As the quantity of biodegradable polymer isdetermined by the size of the microspheres if the concentration of thebiodegradable polymer dissolved in the biodegradable polymer phasesolution is set to be a specific value or a specific range, themicrospheres can be designed to have a longer biodegradation period bymaking them to have a larger size. In other word, the size of themicrospheres may be determined according to the desired biodegradationperiod.

However, if the size of the microspheres is under 20 μm, macrophages maypredate the microspheres and the biodegradation period may consequentlybe shortened. Therefore, the three exemplary medical products shouldhave a target size greater than 20 μm and at the same time, should besmall enough to be injected. The injectability issue will be discussedlater.

The drug delivery rate, by definition, is the degree of absorption intothe body per unit time of the drug dissolved in the biodegradablepolymer making up the microspheres or the rate at which the drugdissolved in the biodegradable polymer making up the microspheres getabsorbed into the body. The drug delivery rate corresponds to thesurface occupancy area or surface occupancy ratio of the drug in themicrospheres, the implication thereof being that it depends mainly onthe concentration ratio between the biodegradable polymer and drugconstituting the microspheres. That is, the size of the microspheres isrelated to the biodegradation period or the release period of the drugand the relevance thereof to the drug delivery rate is comparably small.The injectability refers to the ease of dermal injection using thesyringe and the presence of pain or foreign body sensation at the timeof injection or after injection. Injectability is an especiallyimportant factor when the medical products incorporating therein thebiodegradable polymer-based microspheres are used as injectable medicalfillers or in injectable drug delivery systems.

The smaller the needle used for the infusion of microspherical products,the less pain the person feels. Therefore, the smaller the size of themicrospheres, the smaller the inner diameter of the needle can be used,allowing the microspheres to pass through the needle without cloggingand penetrate into the skin. As the size of the microspheres increases,it is necessary to use a needle having a larger inner diameter, and theperson being injected may feel the pain or discomfort due to theinjection and may also feel a greater foreign body sensation after theinjection.

However, as described above, since the size of the microspheres isstrongly related to the biodegradation period, the size of themicrospheres should be appropriately selected depending on the subjectto which these products are to be applied and the purpose of usethereof.

For example, if these medical products are injected into the human asmedical fillers and hair loss preventives, usually multiple injectionsare required to be performed on the face or scalp. Therefore, whenapplying these products as medical fillers and hair loss preventives,injectability should be the primary consideration. For such applicationsrequiring multiple injections into the human, the microspheres in theseproducts should be of as small a size as possible with an adequatebiodegradation period, and hence, for these applications, the diameterof the microspheres in the medical products ranges from 20 μm to 70 μm,for example, usually around 50 μm.

On the other hand, when these products are injected into animals as adrug delivery system (DDS), the frequency and the site of injection arenot as critical. Such example includes Heartworm preventives, which isusually injected into the chest of a dog and the injectability may notbe as critical as for the human. For such applications requiringinjection into animal skins are desired, microspheres in the medicalproducts may have a elatively large diameter with a view to obtaining aslong a biodegradation period as possible. The diameter of themicrospheres in such cases may range from 100 μm to 150 μm, for example,be around 130 μm.

Based on these considerations, the inventors have applied the basicprinciples for optimization of the mass production of microsphere andthe apparatus based thereon in the production of three types of medicalproducts incorporating therein biodegradable polymer-based microspheresin two size ranges, i.e., 20 μm to 70 μm and 80 μm to 130 μm. That is,the microchannels in the mulitchannel microsphere forming unit aredesigned to have one side thereof being 50 μm and 110 μm, respectively,wherein the multichannel microsphere forming units having microchannelswith one side thereof being 50 μm used for the mass production of themicrospheres having a diameter in 20 μm to 70 μm range and the other,i.e., the multichannel microsphere forming unit including thereinmicrochannels with one side thereof being 110 μm for the mass productionof microspheres having a diameter in 80 μm to 130 μm range, based on the30% rule for the microchannel dimension required described hereinabove.

Once the dimension of the microchannels is fixed, the flow rate of thewater-phase solution is then used to further control the diameter of themicrospheres formed based on the basic principles, followed by using theflow rate of the biodegradable polymer-phase solution to fine tune thediameter thereof.

The flow rate of the water-phase solution and the flow rate of thebiodegradable polymer-phase solution are controlled by controlling theflow rates of the solutions supplied to the multichannel microsphereforming unit 100 from the first reservoir 300 and the second reservoir400 using the flow rate control unit 200.

These design techniques are incorporated in the mass productionapparatus developed and described above to allow the fabrication of thedesired medical products.

The modification incorporated into the mass production apparatusdepending on the application requirement resulted in the production of alarge volume of monodisperse biodegradable polymer-based microsphereshaving the desired target size.

Further investigation of the three medical products fabricated using theprocesses and the mass production apparatus of the present invention isas follow.

Microspherical Products Developed for Medical Filler

As a method for removing wrinkles, medical fillers, such as injectablebovine collagen, have been injected locally into the wrinkled facialarea.

Such a medical filler, however, needs to be improved in the followingareas: (1) must have a close dimensional accuracy with narrow sizedistribution to reduce the side effects such as allergies caused by thebio-toxicity and Kreutzfeld Jacob's disease; (2) should preferably beable to prevent the absorption in vivo for increased sustainability; and(3) should preferably be able to guarantee dermal injectability. As aconsequence, there has been a growing need for the development of amedical filler that does not cause any undesirable biological reactionsin the human body, does not cause aggregation, needle clogging, ornodule forming during injection, and shows slow reabsorption, and onetype of medical fillers meeting the requirements described is a medicalfiller incorporating therein biodegradable polymer-based microspheres,for example is ELLANSE™ M.

Generally, biodegradable polymer-based fillers have been prepared usingphase separation, spray drying, and solvent extraction-evaporation.These manufacturing methods tend to manufacture microspheres having arelatively broad size distribution and a non-uniform size. Therefore, toobtain microspheres of the desired size, a separate size-refiningprocess, such as sieving is required. As a result, microspheres rangingoutside the target size are discarded, resulting in a decrease in finalyield.

Thus, the inventors of the present invention have investigated the massproduction of monodisperse biodegradable polymer-based microspheres,which slowly degrade inside the human body, using the basic principlesdeveloped and the mass production apparatus based thereon with a view toapplying and extending in the technology developed in the massproduction of PDDS for various purposes.

Monodisperse biodegradable polymer-based microspheres for use in medicalfillers having a diameter close to the target diameter determined basedon injectability, reducing needle clogging, nodule formation andflocculation and finally, reducing the occurrence of foreign bodysensation or pain at the time of injection or after the injection aremass produced using the basic principles and the mass productionapparatus based thereon.

The mass produced biodegradable polymer-based microspheres for use inmedical fillers using the basic principles and the apparatus of thepresent invention are monodispersed and have a spherical shape ingeneral, the diameter of the microspheres being about 20 μm to 70 μm,depending on the target initially set.

Further, the size distribution (span value) of the biodegradablepolymer-based microspheres is one or less according to the formulabelow:

$\frac{{Dv}_{0.9} - {Dv}_{0.1}}{{Dv}_{0.5}}$

wherein, Dv_(0.1) is the size of microsphere within 10% in distribution,Dv_0.5 is the size of microsphere within 50% in distribution, and Dv_0.9is the size of microsphere within 90% in distribution. If the sizedistribution is larger than one, the size distribution is regarded asuneven, and the yield of the microspheres will be low.

In one embodiment of the present invention, the biodegradable polymer ofthe present invention is selected from the group consisting ofpolylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolicacid(PLGA), polycaprolactone (PCL), and derivatives thereof, and ispreferably polycaprolactone, but is not limited to the examples. Thenumber average molecular weight of the biodegradable polymer is notparticularly limited, but is 5,000 to 300,000, preferably 8,000 to250,000, and more preferably 10,000 to 200,000.

In one embodiment of the present invention, the microspheres comprisingthe biodegradable polymer produced using the production method andapparatus of the present invention are not particularly limited in theiruse, but may be, for example, a skin cosmetic or medical filler, and canbe used as a subcutaneous or intradermal injection-type filler,particularly in vivo, but is not limited to the examples.

In one embodiment of the present invention, the in vivo resorption timeof the biodegradable polymer-based microspheres produced according tothe production method and the apparatus of the present invention is notparticularly limited, but considering the use as biodegradable skincosmetics or medical fillers, it is preferable that they are resorbed invivo within one to three years.

[Product Example 1]—Mass Production of Monodisperse BiodegradablePolymer-Based Microspheres

Considering the biodegradation period and injectability of thebiodegradable polymer-based microspheres for use in medical fillers, theoptimum size of the microspheres needed was determined to be 60 μm, withthe lower limit and the upper limit being 40 μm and 80 μm, respectively.

The biodegradable polymer-phase solution was prepared by dissolving 15wt % of Polycaprolactone (PCL) having a Mn of 45,000 in dichloromethanesolvent (boiling point: 39.6° C.) and the water-phase solution, bydissolving, as a surfactant, 0.25 wt % of Polyvinyl alcohol (PVA) havinga molecular weight of 85,000 to 124,000 in purified water. One side,i.e., the width, of the microchannel incorporated in the multichannelmicrosphere forming unit 100 of the mass production apparatus inaccordance with the present invention was set at 60 μm.

The water-phase solution and the biodegradable polymer-phase solutionwere filled respectively into the first reservoir 300 (see FIG. 13) andthe second reservoir 400 (see FIG. 14) before being fed into themultichannel microsphere forming unit 100 of the mass productionapparatus illustrated in FIG. 16.

At this time, the flow rate of the aqueous solution was increased ordecreased while the flow rate of the biodegradable polymer phasesolution was kept constant, and the size of the microspheres formed wasdetermined. The flow rate of the aqueous solution was then adjusted sothat the microspheres were in the vicinity of the target size, that is,about 50 μm.

Then, the size of the microspheres formed was fine-tuned by adjustingthe flow rate of the biodegradable polymer phase solution while the flowrate of the aqueous solution was kept constant.

Referring to FIG. 16, the flow rate of water-phase solution and the flowrate of the biodegradable polymer-phase solution in the multichannelforming unit 100 are adjusted using the flow rate control unit 200.

The temperature of the water-phase solution and the biodegradablepolymer-phase solution were maintained at 15° C.

The microspheres in the product reservoir 600 (see FIG. 16) werecollected. Thereafter, the solvent, e.g., dichloromethane, was firstextracted from the collected microspheres.

After filtering the water-phase solution containing the microspheres,the residual surfactant and solvent, e.g., PVA and dichloromethane, wereremoved from the microspheres through washing before being dried,resulting in the formation of the desired monodisperse biodegradablepolymer-based microspheres.

[Product Example 2]—Preparation of Monodisperse BiodegradablePolymer-Based Microspheres for Use in Medical Fillers

Since the minimum diameter of the microspheres for being injected in tothe human body is between 20 and 30 μm, the target diameter of themicrospheres for use in medical fillers to be formed using the basicprinciples and the mass production apparatus developed based thereon wasset between 20 μm and 40 μm, and in line with the basic principles,i.e., so-called the 30% rule, one side, i.e., the width, at least, ofthe microchannels in the multichannel microsphere forming unit 100 wasset at 30 μm.

Other than the dimension of the microchannels, the same processesdescribed in the Product Example 1 were used to prepare the monodispersebiodegradable polymer-based microspheres for use in medical fillers.

[Comparative Example]—Formation of PCL-Based Microspheres Using thePrinciples and the Apparatus Developed and Comparison Thereof with thePCL-Based Microspheres in ELLANSE™ M Formed Using the SolventExtraction-Evaporation

The biodegradable polymer-based microspheres used in ELLANSE™ M, acommercially available and representative medical filler, are preparedusing the solvent extraction-evaporation method and are used as acomparison, i.e., as the Control Product.

The biodegradable polymer-phase solution was prepared by dissolvingpolycaprolactone (PCL) having a Mn of 45,000 in a solvent ofdichloromethane (boiling point: 39.6° C.) at a concentration of 15 wt %and the water-phase solution, by dissolving polyvinyl alcohol (PVA)having a Mw of 85,000-124,000 as a surfactant in purified water at aconcentration of 0.25 wt %.

The desired monodisperse biodegradable polymer-based microspheres wereformed by introducing these solutions into the mass production apparatusof the present invention and manipulating the process parameters asdescribed hereinabove.

[Test Example 1] SEM Image Analysis of the Microspheres for Use inMedical Fillers

The monodisperse biodegradable polymer-based microspheres obtained usingthe basic principles and the apparatus developed in Product Example 1were visually investigated. There are shown in FIGS. 8 and 9 a SEM imageof single biodegradable polymer-based microsphere obtained and a SEMimage of monodisperse biodegradable polymer-based microspheres obtained,respectively, each of the microspheres having a diameter of 50 μm.

As shown in FIGS. 8 and 9, the mass-produced microspheres of ProductExample 1 are spherical with a smooth surface and are monodispersed,each of the microspheres having a constant size and shape.

There are showin FIG. 56 a comparison of the SEM images of themicrospheres produced in Product Example 2, each of the microsphereshaving a target dimeter of 30 μm and those for ELLANSE™ M having adiameter about 40 μm, as the Control Product.

Using a SEM, the morphology of microspheres was visually compared withthat of the commercially available product, ELLANSE™ M. According to themorphology analysis, the morphology of the microspheres of ProductExample 2 is comparable to that of ELLANSE™ M, i.e., spherical in shape.

It is relatively harder to produce monodisperse microspheres with asmaller diameter than the ones with a large diameter. Product Example 2has a smaller diameter than Product Example 1 and Comparative Example,that is, ELLANSE™ M. In spite the microspheres of Product Example 2mass-produced using the basic principles and the mass productionapparatus according to the present invention are smaller in size, theyhave a smoother shape than the Comparative Example/Control Agent, i.e.,ELLANSE™ M, and the overall size distribution is relatively constant andmonodispersed.

In particular, the production of the biodegradable microspheres used inELLANSE™ M using the solvent extraction-evaporation method, results alow yield, for the solvent extraction-evaporation method results inproducing microspheres having a wide particle size distribution and inorder for the biodegradable microspheres to be used in the product,those microspheres not meeting the size requirement must first beselectively filtered and sieved out, leaving only a small amount of themicrospheres from the lot meeting the size requirement.

However, in contrast to the solvent extraction-evaporation method, thebasic principles and the mass production apparatus of the presentinvention are capable of ensuring a mass production of monodispersebiodegradable microspheres from the beginning of the production,therefore, not requiring the filtering and sieving steps required in thesolvent extraction-evaporation method, minimizing the loss therefrom,which, in turn, results in a high yield.

[Test Example 2] GPC Analysis of the Microspheres for Use in MedicalFillers

Since the biodegradation period of the microsphere is dependent on themolecular weight of the polymer, the molecular weight of themicrospheres of Product Example 2 and that of the Control Agent wasinvestigated.

There are shown in FIG. 57A to C the results of GPC analysis of the rawpowder of the biodegradable polymer PCL (Polycaprolactone, PURAC®) used,the results of GPC analysis of the Control Product, i.e., ELLANSE™ M,and the results of GPC analysis of the Test Product, i.e., microspheresof Product Example 2. Table 7 summarizes the results of GPC analysis ofraw powder for biodegradable polymers, the Control Product, and TestProduct.

TABLE 7 Results of GPC analysis of raw powder for biodegradablepolymers, the Control Product, and Test Product Poly Classification MnMw MP Mz Mz + 1 Dispersity Material PCL(PURAC ®) 14461 23421 23738 3375043427 1.619576 (Powder) Particle control agent 15013 23829 22672 3377643344 1.587199 (ELLANSE ™ M) Test Agent 13259 23032 21825 34233 447181.737021 (product example 2)wherein,

a) Mn is Number-average Molecular Weight

b) Mw is the weight-average molecular weightc) Mz is the Z-average molecular weight.

According to the results obtained from the GPC analysis, the molecularweights of the PCL powder as the raw material, the Control Product,i.e., ELLANSE™ M and the Test Product, i.e., the microspheres of ProductExample 2 were similar to each other and the based thereon, it can beconcluded that the Control Product and the Test Product have the similarbiodegradation period and size distribution as the polymer concentrationtherein varies.

[Test Example 3]—Size Distribution Analysis of the Microspheres for Usein Medical Fillers

The diameters of the Control Product, i.e.,ELLANSE™ M, and the TestProduct, i.e., the microspheres of Product Example 2, were compared andanalyzed using a particle size analyzer (PSA).

FIG. 58A is a graph of the particle size distribution of the ControlProduct, i.e.,ELLANSE™ M, and FIG. 58B, a graph of the particle sizedistribution of the Test Product, i.e., the microspheres of ProductExample 2.

Referring to FIG. 58A, the Control Product, i.e.,ELLANSE™ M, wasanalyzed to have an average diameter of 41.59 μm and was analyzed tohave a size distribution of 19.95 μm.

Referring to FIG. 58B, the Test Product, i.e., microspheres of ProductExample 2, was analyzed to have an average diameter of 33 μm and wasanalyzed to have a size distribution of 16.65 μm.

As described above, the microspheres of Product Example 2 have a size of30 μm, close to 20 μm, which is close to the limit diameter forexhibiting an appropriate biodegradability in vivo. Despite thedifficulty of controlling the size distribution when the microspheresare small, the Test Product, i.e., Product Example 2, had a narrowerdiameter distribution, i.e., monodisperse, and also a smaller diameterthan the Control Product, i.e.,ELLANSE™ M.

[Test Example 4]—Viscoelasticity of the Microspheres for Use in MedicalFillers

The viscoelastic properties of the Test Product, i.e., the microspheresof Product Example 2, and the Control Product, i.e., ELLANSE™ M weremeasured and compared using Rheometer (Kinexus, Malvern, U.K.). Inaddition, as other Control Products, the viscoelastic properties ofRESTYLANE® (Perlane+Lido) and AESTHEFILL® (V200) were also measured andcompared.

There are shown in FIG. 59A to D comparisons of the measured elasticity,the measured viscosity, the measured complex viscosity and the phaseangle of the Test Product and the Control Products, respectively. Table10 summarizes the viscoelastic measurements of the Test Product andControl Products.

TABLE 10 Summary of the Viscoelastic Measurements of the Test Productand Control Products Storage Loss Complex Phase Modulus¹ Modulus²Viscosity³ Angle⁴ G′(Pa) G″(Pa) η*(Pas) δ(°) Test Product 573.9 257.4100.3 24.16 RESTYLANE ® 566.2 105.2 91.88 10.53 Perlane + LidoAESTHEFILL ® 14.4 21.85 4.175 56.62 V200 ELLANSÉ ™ M 537.4 436.5 110.439.08wherein, G′ is the storage modulus, i.e., the elastic portion of thesample at the measured frequency. G″ is the loss modulus, i.e., theviscous portion of the sample at the measured frequency. η* (complexviscosity) is a vector term representing viscosity and elasticitytogether that reflects the degree of deformation against external forceand δ is the phase angle between viscosity and elasticity terms of η*.

Compared with the Control Product (ELLANSE™ M), the elasticity of theTest Product (Product Example 2) was similar to that of the ControlProduct, but the viscosity of the Test Product was lower than that ofthe Control Product. The smaller the viscosity of the microspheres is,the more easily the microspheres can be injected into the human body. Inother words, since the viscosity of the Test Product is lower viscositythan that of the Control Product (ELLANSE™ M), the Test Product can beinjected more easily into the human body. Further, since the elasticityof the test product is similar to that of the Control Product (ELLANSE™M), the supporting force of the Test Product is as good as that of theControl Product in the human body, allowing the microspheres of the TestProduct to physically maintain themselves as good as the Control Productin the human body.

In addition, according to the measured complex viscosities, the degreeof deformation by the external force was analyzed to be similar in theTest product and Control Product (ELLANSE™ M).

[Test Example 5]—Injection Force Analysis of the Microspheres for Use inMedical Fillers

Medical fillers should be injected into the human body without anexcessive force. Thus, the Test Product, i.e., the microspheres ofProduct Example 2, and the Control Product, i.e., ELLANSE™ M, werecompared with each other using a texture analyzer (Stable Micro System,U.K.). Additionly, the injection forces for other Control Products suchas RESTYLANE® (Perlane+Lido) and AESTHEFILL® (V200) were also measuredand compared.

FIG. 60A is a graph of the injection force measurements of the Test andControl Products obtained using a TERUMO 27Gx3/4 needle and FIG. 60B isa graph of the injection force measurements of the Test and ControlProducts obtained using a DN Co. 27G needle. Table 11 summarizes theinjection force measurements of the Test and Control Products above.

TABLE 11 the injection force measurements of the Test and ControlProducts Test Product RESTYLANE ® AESTHEFILL ® (Example Product 2)Perlane + Lido V200 ELLANSE ™ M TERUMO DN Co. TERUMO DN Co. TERUMO DNCo. TERUMO DN Co. 27G × ¾ 27G 27G × ¾ 27G 27G × ¾ 27G 27G × ¾ 27G 0.4 ×20 mm 38 mm 0.4 × 20 mm 38mm 0.4 × 20 mm 38 mm 0.4 × 20 mm 38 mm  5mm/min 13.50 16.52 22.68 16.09 7.58 10.32 26.88 35.38 15 mm/min 18.0721.15 23.53 24.98 10.72 13.82 28.57 39.66 30 mm/min 24.50 25.90 29.5726.27 13.36 17.62 32.03 41.66

The measurement results show that the Test Product can be injected underthe skin with a less force than the Control Product (ELLANSE™ M), i.e.,the test product requires less injection force and provides a moreconsistent control of the injection force than the Control Product(ELLANSE™ M).

[Test Example 6] Durability & Migration Test of the Microspheres for Usein Medical Fillers

Medical fillers should preferably be provided with a sufficientdurability to maintain the supporting force after being injected underthe skin. Thus, the volume change of the Test Product, i.e., themicrospheres of Product Example 2 and the Control Product, i.e.,ELLANSE™ M, against the time was measured and compared using PRIMOSLITE(with Software PRIMOS 5.8). In addition, the volume change against thetime were measured and comapred for other Control Products, i.e.,RESTYLANE® (Perlane+Lido) and AESTHEFILL® (V200) after being injectedunder the skin.

FIG. 61 is a photograph showing the volume changes in the microspheresof Product Example 2, i.e., Product Example 2 (IVL-F) and other ControlProducts against the time after being injected under the skin of thelaboratory mice.

According to the results obtained, the Test Product is provided with alonger intradermal persistence period than the Control Product (ELLANSE™M).

[Test Example 7] Preparation of the Microspheres for Use in MedicalFillers: Biological Stability Evaluation

As a preparation for the clinical test of the medical fillerincorporating threin the microspheres produced, i.e, the microspheres ofProduct Example 2, using the basic principles and the apparatus of thepresent invention, the biological stability evaluation thereof, wascarried out and the results therefrom are summaried in Table 12.

TABLE 12 Results of the Biological Stability Evaluation of the TestProduct Test Items Result Evaluation Test Method Cytotoxicity Grade 0Suitable Test according to ISO Test (Noncytotoxic) 10993-5, CytotoxicityTest (Indirect (Indirect Contact—Agar contact Diffusion Test). method—(Tested with undiluted agar diffusion solution) method) Pyrogenicity NoNZW rabbit under test Suitable ISO 10993-11, Test showed a rise in bodyPyrogenicity Test. temperature by 0.5° C. or (Prepare the sample higherin the pyrogenicity solution: 4 g/20 mL, test, which evaluated thecentrifuge at 3,000 rpm agent as a negative for 10 min at 37° C. for 72pyrogenic substance. h, filter through a 0.45 μm filter and use as thesample solution) Intradermal No NZW rabbit under test Suitable Testaccording to ISO Reaction was observed to have 10993-10 IntracutaneousTest erythema and edema in Reactivity test. intradermal reaction test.Calculated difference between the test and control agents was less than“1.0”. Acute No systemic toxicity Suitable Test according to ISOToxicity Test change among ICR mice 10993-11 Acute systemic under testwas observed toxicity. (Administration within 72 hours after route:abdominal cavity) injection. Genotoxicity Negative Suitable Testaccording to ISO Test 10993-3, Test for (Microbial genotoxicity. ReturnMutation) Genotoxicity Negative Suitable Test according to ISO Test10993-3, Test for (Micronucleus genotoxicity. test) Skin Sensitizationtest results of Suitable Test according to ISO Sensitization this agentagainst Dunkin 10993-10, Maximization Test Hartley guinea pig weresensitization test. (Maximization evaluated as weak in method)sensitization because it did not cause a skin reaction. Subchronic Nosignificant systemic Suitable Test according to ISO Toxicity Testtoxicity change was 10993-11, subchronic including observed in all SDrats test systemic toxicity of Test Transplantation group for 90 daysafter for systemic toxicity and (90 days, transplant compared with ISO10993-6, Test transplant) the control group. The method implantation inhistological evaluation subcutaneous tissue. (90 indices of the testdays, undiluted solution, transplantation site were subcutaneouscalculated as zero in both transplantation) male and female groupscompared with the control group, which evaluated both male and femalegroup as “non-irritant.” Aseptic Test Suitable Suitable Test accordingto Aseptic Test Method in General Test Methods of the KoreanPharmacopoeia.wherein, the infusion solution is in accordance with [Common Standardfor Biological Safety of Medical Devices] or ISO 10993.

Based on the results of the biological stability evaluation, it can beconcluded that the

Test Product has achieved/met all of the required biological stabilityrequirements.

Biodegradable Microspheres Developed for Use in Heartworm Preventives

The Dirofilaria Immitis, also known as Heartworm, is a parasite that istransmitted mainly by mosquitoes and detrimentally affects the heart ofpets such as dogs and cats. When infected, it eadicated with an arsenicagent (caparsolate) or with melarsomine. However, all of the abovetherapeutic agents above have serious side effects such as irritation atthe injection site and liver/kidney damages.

Therefore, it is safe and economical to prevent the Heartworm diseasebefore being infected and Diethylcarbamazine (DEC), Ivermectin,Milbemycin, Moxidectin, Selamectin, etc. are known to prevent theHeartworm disease.

These preventive medicines should be given on a daily-basis or monthlyintervals to ensure adequate preventive effects, but if the prescribedmedication period is not properly kept, the preventive effect maydisappear, exposing the pets to the risk of Heartworm infection.

Thus, the inventors have attempted to mass produce, using the basicprinciples and the apparatus in accordance with the present inventionwhich are ideally suited for the mass production of polymeric DDSs asdescribed above, biodegradable polymer-based microspheres, uniformlydissolved therein one of the Heartworm preventive agents above to beslowly released into the body as the biodegradable polymer in themicrosphere degrades after being injected, similar to how polymeric DDSsfunction in human body.

The microspheres, including therein one of the preventive agents above,for use in Heartworm preventives in accordance with one embodiment ofthe present invention are to be administered through the syringe intothe subject's skin, maintaining the effectiveness for a prolonged periodof three to six months, the period coinciding with the degradationperiod of the biodegradable polymer. In addition, since the massproduction apparatus according to the present invention is capable ofmass producing monodisperse biodegradable polymer-based microspheresdissolved therein one of the Heartworm preventive agent, the Heartwormpreventive incorporating therein the microspheres of the presentinvention, as a consequence of the monodispersity of the microspheres,can maintain the intended drug delivery rate, coinciding with thedegradation rate of the biodegradable polymer and the drug effectivenessperiod constant, coinciding with the polymer degradation period. Inaddition, using the basic principles and the apparatus developed inaccordance with the present invention, the biodegradable polymer-basedmicrospheres for use in Heartworm preventives can be produced at a highyield, as observed for Medical Fillers above.

The microspheres for use in the Heartworm preventive mass produced inaccordance with the present invention for use in Heartworm preventiveare spherical in general and include therein a Heartworm preventiveagent evenly distributed in a biodegradable polymer.

In one embodiment, the average diameter of the microspheres is fromabout 80 μm to about 130 μm. In another embodiment of the presentinvention, since Heartworm preventive incorporating therein themicrospheres is to be injected into an animal such as a dog or a cat,not into a human, the microspherical product for prevention ofDirofilaria Immitis infestation is injected into an animal such as a dogor a cat and is not injected into a human, pain or foreign bodysensation during injection may not be an issue. Accordingly, it isadvantageous to make the biodegradation period of the microspheres foruse in Heartworm preventives as long as possible with a view to carryingout Heartworm preventive function as long as possible, and, hence, themicrospheres for use in Heartworm preventives are relatively be largerthan the microspheres for use in other medical products to be injectedinto the human body.

In one embodiment of the present invention, Moxidectin is used as theHeartworm preventive agent and there is shown in FIG. 62 the chemicalformula and structure thereof.

In one embodiment, the microspheres of the present invention maycomprise a biodegradable polymer and Moxidectin in a weight ratio of 4:1to 9:1, preferably 4:1. When the weight ratio of the biodegradablepolymer and the moxidectin is less than 4:1, the shape-retaining forceof the biodegradable polymer may be weakened, possibly resulting in anuneven distribution of Moxidctin and the biodegradable polymer in themicrospheres. In addition, if the weight ratio of the biodegradablepolymer and the moxidectin exceeds 9:1, a considerable amount ofheartworm preventive should preferably be administered to the subject,which may cause discomfort to the subject.

More specifically, the concentration of the biodegradable polymer in thebiodegradable polymer-phase solution should be between 15 wt % and 60 wt%, preferably 15 wt %, but is not limited to the above example.

In one embodiment, the microspheres of the present invention maycontinuously release Moxidectin in the body for three to six months.

In one embodiment, the biodegradable polymer of the present inventionmay be selected from the group consisting of polylactic acid,polylactide, polylactic-co-glycolic acid, polylactide-co-glycolide(PLGA), polyphosphazene, polyiminocarbonate, polyphosphoester,polyanhydrides, polyorthoesters, polycaprolactones, polyhydroxyvalates,polyhydroxybutyrates, polyamino acid, and combinations thereof.

In one embodiment, the microspheres of the present invention areprepared by using the basic droplet-based HCMMM microspheremanufacturing method described above with reference to FIG. 2, whereinthe mixture is properly mixed/dissolved in the biodegradablepolymer-phase solution.

In another embodiment, the microspherical products of the presentinvention are formed using the mass production apparatus described withreference to FIGS. 16 to 31.

[Manufacturing Example]—Preparation of Microspheres Including Moxidectinfor Use in Heartworm Preventives

The diameter of the microspheres for use in Heartworm preventive to besubcutaneously injected into the animal is set between 80 μm and 130 μm,preferably set to be 100 μm, considering the biodegradation period, drugdelivery rate and injectability of the Heartworm preventive includingtherein the microspheres, and the concentration of biodegradable polymerand Heartworm preventive agent, i.e., moxidectin, was set as follows.

The biodegradable polymer phase solution was prepared by dissolvingpolylactide-co-glycolide (PLGA) and Moxidectin in an organic solvent,i.e., dichloromethane. The concentration of the biodegradable polymer,i.e., PLGA, in the biodegradable polymer-phase solution was set at 15 wt% and the weight ratio of PLA to Moxidectin was set at 4:1.

The water-phase solution was prepared by dissolving Polyvinyl alcohol(PVA) as a surfactant in pure water with water at a concentration of0.25 wt %.

In one of the side, e.g., the width, of the microchannels in themultichannel microsphere forming unit 100 was set at 100 μm.

The water-phase solution and biodegradable polymer-phase solution wereintroduced into the microchannels formed on a silicon wafer. The flowrate of the water-phase solution was adjusted while holding the flowrate of the biodegradable polymer-phase solution constant until thediameter of the microspheres formed reaches the target diameter, i.e.,100 μm.

Then, the flow rate of the biodegradable polymer-phase solution wasadjusted while holding the flow rate of the water-phase solutionconstant to fine-tune the diamter of the microspheres obtained from theabove described precedure.

Referring to FIG. 16, the flow rate of the water-phase solution and theflow rate of the biodegradable polymer-phase solution are adjusted bycontrolling the flows from the first reservoir 300 and the secondreservoir 400 to the multichannel microsphere forming unit 100 using theflow rate control unit 200.

The water-phase solution and biodegradable polymer-phase solution weremaintained at a temperature of 15° C.

The microspheres formed in the product reservoir 600 (see FIG. 16) werecollected to be stirred. There are three stirring stages. The firststage stirring was carried out at a speed of 300 rpm for 1 hour at 17°C., followed by the second stage stirring, at a temperature of to 25°C., at a speed of 400 rpm for 1 hour and finally, the third stagestirring, a temperature of 45° C. at a speed of 500 rpm for 4 hours.

The desired microspheres were obtained by washing several times themicrospheres that underwent the three stirring stages with purifiedwater and by lyophilizing the microspheres that underwent the washingstages.

[Comparative Example]—Heartworm Preventives

As a comparative example, a commercial avialble Heartworm preventive,namely, Proheart® SR-12 was used.

[Test Example 1] SEM Image Analysis of the Microspheres for Use inHeartworm Preventives

The morphology of the Control Product, i.e., commercially availableProheart®, and the Test Product, i.e., the microspheres for use inHeartworm Preventives in accordance were analyzed and compared using SEMimages thereof.

FIG. 63 is a comparison of the SEM images of the microspheres for use inHeartworm Preventives manufactured using the mass production apparatusand the basic principles in according with the present invention, i.e.,the Test Product, and FIG. 63 is the SEM images of the microspheresincorporated in the Control Product, i.e., Proheart®.

Based on the analysis of SEM images of the microspheres of the TestProduct, i.e., the microspheres obtained using the principles and themass production apparatus of the present invention, and the microspheresincorporated in the Control Product, i.e., PROHEART®, it can beconcluded that the microspheres of Test Product have a spherical shapecomparable to that of the Control Product.

Further, the microspheres of the Test Product have a smoother shape thanthe microspheres of the Control Product and the overall sizedistribution thereof is relatively constant, i.e., are monodispersed.

[Test Example 2]-Size Distribution Analysis of the Microspheres of theTest Product and the Control Product

The diameters of the microspheres of the Control Product, i.e.,PROHEART® and the Test Product, were compared and analyzed using aparticle size analyzer (PSA).

FIG. 64 is a graph illustrating the results of the particle sizeanalysis of the microspheres of the Test Product and the Control Productand Table 12 is a summary thereof.

According to the analysis, the Test Product was analyzed to have asmaller mean diameter and also a narrower distribution compared to theControl Product, implying that the Test Product can be injected into theanimal using a high G (gage) needle having a narrower inner diameter,i.e., a higher injectability compared to the Control Product.

TABLE 12 Summary of the Size Distribution Analysis of the Microspheresof the Test Product and the Control Product X₁₀ X₅₀ X₉₀ SMD VMD Product(μm) (μm) (μm) (μm) (μm) Control 110.50 132.48 148.62 130.39 131.91Product (PROHEART ® SR-12) Test Product 98.09 116.93 140.77 99.37 116.01

[Test Example 3]—Content Test of the Microspheres for Use in HeartwormPreventives

Table 13 describes the test items and methods used for the content test.The Test Product was tested for the contents according to the procedureas described in Table 13.

TABLE 13 Content Test Method Standard 0.1 g of Standard Moxidectin wasdissolved in Solution Acetonitrile to obtain 10 ml of AcetonitrileSolution. The Standard Solution was obtained by adding acetonitrile to 1ml of Acetonitrile Solution until the volume thereof reaches 10 ml. TestDissolve 1 g of the microspheres in Acetonitrile until Solution thevolume reaches 10 ml. Use this solution as the Test Solution. FormulaPeak Area of the Test Solution/Peak Area of the Standard Solution ×Content of Standard Moxidectin × 100

The content was calculated as a product of the Test Solution Peak Areadivided by the Standard Solution Peak Area and multiplied by the Contentof Standard Moxidectin multiplied and by 100.

According to the results of the test, the content of Moxidectin in theTest Product was measured to be 96.26% (±0.25%), meaning that theMoxidectin content of the Test Product was analyzed to be within thenormal drug content range of 95.0 to 105.0%.

[Test Example 4]—Release Test of the Microspheres for Use in HeartwormPreventives

Table 14 describes the test items and methods for the release test ofthe microspheres for use in Heartworm Preventives. The release test ofthe Test Product was carried out according to the procedure as describedin Table 14

TABLE 14 Release Test Method Release 0.9% SLS Test solution Release 1.Fill 100 mg of this product in a 120 ml glass Test Method test containerfilled with 100 ml of the release test solution 2. Place it in a 37° C.water bath and shake it with a vibration amplitude 4 cm and a shakingfrequency 120 times/minute (reciprocation) 3. Shake well before taking 1ml of specimens when collecting. 4. Run centrifuge at 13,000 rpm for 3minutes, take the supernatant and analyze by HPLC (Analyze by HPLC afterfiltering using 0.45 μm PTEF syringe filter) Release 37° C. TemperaturePaddle rpm 120 rpm Sampling 15, 30 min, 1, 2, 4, 8 hr, 1, 2, 3, 4, 7,10, 14 day Time

There are shown in FIGS. 65A and B the result of the in vivo releasetest of the control product (PROHEART® SR-12) and that of the TestProduct, respectively.

According to the body release test, the total amount of drug released inthe body increased with time in the Control Product and the TestProduct, and both showed similar overall results.

In this graphs, the drug delivery rate will be the slope of the drugrelease over time. The Control Product showed a decrease in drugdelivery rate near the final release stage, whereas the Test Product wasfound to maintain a constant slope, that is, a constant drug deliveryrate even after a long period of release, showing a clear advantage overthe Control Product.

[Test Example 5] Morphology Variation of the Microspheres for Use inHeartworm Preventives with Respect to the Concentration of theBiodegradable Polymer (PLA) Therein

The morphological changes of the microspheres were observed as theconcentration of the biodegradable polymer (PLA) therein was varied,from 40 wt % to 50 wt % and finally to 60 wt %.

There are shown in FIG. 66A to C an SEM image when the concentration ofthe biodegradable polymer (PLA) is 40 wt %, when the concentration ofthe biodegradable polymer is 50 wt % and when the concentration of thebiodegradable polymer is 60 wt %, respectively.

It was confirmed through the observation that as the concentration ofthe biodegradable polymer, which functions as the skeleton in themicrospheres incorporating therein Moxidectin and biodegradable polymer,increases, microspheres having more perfect spherical shape and asmoother surface are obtained.

[Test Example 6]—Analysis of the Particle Size Distribution of theMicrospheres for Use in Heartworm Preventives as the Concentration ofthe Biodegradable Polymer (PLA) is Varied

The particle size distribution of the microspheres of the Test Productwere analyzed using a particle size analyzer (PSA) as the concentrationof the biodegradable polymer (PLA) therein were varied, from 40 wt % to50 wt % and finally to 60 wt %.

There are shown in FIG. 67A to C, a graph of the particle size analysiswhen the concentration of the biodegradable polymer of the Test Productis 40 wt %, when the concentration of the biodegradable polymer of theTest Product is 50 wt % and when the concentration of the biodegradablepolymer of the test product is 60 wt %, respectively.

Referring to FIG. 67A, the Test Product had an average diameter of 133.2μm and was analyzed to have a diameter distribution of 56.43 μm centeredon the average diameter.

Referring to FIG. 67B, the Test Product had an average diameter of 125.3μm and was analyzed to have a diameter distribution of 55.77 μm centeredon the mean diameter.

Referring to FIG. 67C, the test product has an average diameter of 96.69μm and was analyzed to have a diameter distribution of 50.97 μm centeredon the mean diameter.

According to the results of the particle size distribution analysis, thediameter of the prepared microspheres decreased as the concentration ofthe biodegradable polymer increased, and was analyzed to have a narrowdiameter distribution in the range of 50 μm to 60 μm.

[Test Example 7]—Drug Release Test for the Microspheres for Use inHeartworm Preventives as the Concentration of the Biodegradable Polymer(PLA/PLGA) Therein is Varied

The in vivo drug release test for the microspheres was carried out theconcentration of the biodegradable polymer (PLA) of the Test Product wasvaried from 40 wt %, 50 wt % and finally, 60 wt %.

There are shown in FIG. 68A to C, a graph of the drug release testresults when the concentration of the biodegradable polymer of the TestProduct is 40 wt %, when the concentration of the biodegradable polymerof the Test Product is 50 wt % and when the concentration of thebiodegradable polymer of the test product is 60 wt %, respectively.

According to the drug release test results of the microspheres as theconcentration of the biodegradable polymer therein was varied, theconcentration of biodegradable polymer in the microspheres has arelatively small effect on the drug release. If a different drug releaseprofile is desired, a different type or combination of the biodegradablepolymers may be attempted.

Thus, microspheres incorporating therein PLGA (DL-lactide/glycolidecopolymer, PURAC®), which is based on PLA (poly (DL-lactide), PURAC®)polymer with glycolide added thereto to shorten the polymer degradationperiod, was further tested.

[Test Example 8] Morphology Variation in the Microspheres for Use inHeartworm Preventives According to the Mixing Ratio of the BiodegradablePolymer (PLGA) to Moxidectin

The morphological changes in the microspheres were observed as themixing ratio of moxidectin to the biodegradable polymer (PLGA) wasvaried from 1:9, to 1:6 and, finally to 1:4.

There are shown in FIG. 69A to C an SEM image of the microspheres whenthe ratio of moxidectin to the biodegradable polymer (PLGA) is 1:9, thesame when the ratio of moxidectin to the biodegradable polymer (PLGA) is1:6, and the same when the ratio of moxidectin to the biodegradablepolymer (PLGA) is 1:4, respectively.

According to the SEM images of the microspheres incorporating thereinPLGA as the biodegradable polymer, density of the microspheres decreasedas the ratio of the heartworm preventive agent/drug to PLGA polymerincreased, and the rate or amount of drug release should increase as thedensity of the microspheres decreases.

[Test Example 9] In-Vivo Drug Release Test of the Micropsheres for Usein Heartworm Preventives as the Mixing Ratio of the BiodegradablePolymer (PLGA) to Moxidectin Changes

The in vivo drug release tests were performed the microspheres to beused in Heartworm Preventives as the ratio of the heartworm preventiveagent/drug, i.e., moxidectin, to the biodegradable polymer (PLGA) isvaried from 1:9, to 1:6 and finally to 1:4.

FIG. 70 is a graph of the in vivo drug release results of the TestProduct while varying the mixing ratios of the heartworm preventiveagent/drug, i.e., moxidectin, and the biodegradable polymer (PLGA).

Compared with the Control Product, the Test Products having a variety ofmixing ratio of moxidectin and biodegradable polymer (PLGA) haveexhibited shorter in vivo release periods and smaller release amount.However, this is due to the biodegradation period of the Test Productwas designed to be short, and it was confirmed that the drug releaseamount can be increased by increasing the mixing ratio of the heartwormpreventive agent/drug, i.e., moxidectin, to the biodegradable polymer,i.e., PLGA.

[Test Example 10]—Test of the Heartworm Preventive DevelopedIncorporating Therein the Microspheres of the Present Invention onAnimals

Testing of the Test Product was carried out on animals. As the firststep, a test plan was prepared.

The purpose of the animal testing is for the pharmacokinetic evaluationof the Test Product, that is, the mass-produced microspheres for use inHeartworm Preventives in accordance with the present invention and theControl Product, i.e., PROHEART® SR-12.

Pharmacokinetic parameters of moxidectin are provided in Table 15 below.

TABLE 15 Pharmacokinetic Parameter of Moxidectin PharmacokineticParameters Value AUC 217 ng · day/mL Cmax 5.1 ng Tmax 7-10 day T1/2 35day

The animal testing method was designed as follows:

-   1. Blood collections are conducted at 0, 1, 2, 6, 12 hours, 1, 3, 6,    7, 8, 9, 10, 14, 21, 28, 42, 56, 70 and 90 days;-   2. Three groups are tested, each group consisting three specimen;    and-   3. Test group is composed as shown in the following Table 16.-   4. The dosage was set based on the dosage (0.5 mg/kg) of the    commercial Control Product (PROHEART® SR-12). The dosage of the Test    Product was set at ¼ of the Control Product (0.125 mg/kg)    proportional to the expected duration of the drug in the body    (Control Product: 12-month release period and the Test Product:    3-month release period); and-   5. The Control Product and the Test Product were administered once    by subcutaneous injection, and the blood was collected from the    jugular vein and analyzed for the concentration of Moxidectin in the    blood using LC-MS/MS.

TABLE 16 Configuration of the Test Group Number of Animal Group GenderAnimals Number Dose Drug G1 M 3 1-3 0.125 mg/kg Moxi-1Q G2 M 3 4-6  0.5mg/kg PROHEART SR-12 G3 M 3 7-9 2.5 mL Advocate (10-20 kg test groupsetting)wherein, Moxi-1Q refers to the Test Product.

There are illustrated in FIGS. 71A to C a graph of the drug releaseresults of the Test Product, a graph of the drug release results of theControl Product (PROHEART® SR-12) and a combined graph of the drugrelease results of the Test Product and the Control Product.

According to the drug release results, the primary PK test of theControl Product (PROHEART® SR-12) and the Test Product showed a slightdifference in blood concentration. Since, however, these differences canbe overcome by varying the dosage of the heartworm preventive or byincreasing the concentration of the preventive agent/drug therein, itcan be concluded that the Test Product has a drug efficacy comparable tothat of the Control Product.

Microspherical Products Developed for Hair Loss Preventives

There are two types of commercially available oral therapeutic agentsfor preventing hair loss, namely, finasteride or dutasteride. These hairloss treatments using these therapeutic agents, as shown in FIG. 72,inhibit 5-α-reductase inhibitor which acts to convert testosterone intodihydrotestosterone (DHT), which is a strong male hormone, therebyinhibiting DHT production, which, in turn, inhibits the shrinking ofhair root by DHT at the scalp, thereby treating androgenetic hair loss.

5-α-reductase inhibitors can be divided into Type 1 and Type 2. Type 1is found in the scalp and sebaceous glands, and Type 2 in the scalp andprostate. Finasteride only blocks Type 2 of the 5-α-reductase inhibitor,but dutasteride blocks both Type 1 and Type 2 inhibitors. It is knownthat the dutasteride is more potent in inhibiting DHT than finasteride.However, based on the first year of use, the rate of side effects ofdutasteride was higher, and as a consequence, finasteride is now themost widely used and safe treatment for the hair loss, and is the onlyFDA approved drug therefor.

Such oral hair loss treatment drugs can only be effective when taken ona daily-basis for more than 3 months, making it extremely inconvenientfor the patient.

The inventors have investigated the possibility of mass producingbiodegradable polymer-based microspheres incorporating therein the hairloss preventive/therapeutic agent/drug using the basic principles andthe apparatus developed in accordance with the present invention,capable of providing a controlled release of the hair loss therapeuticagent/drug in the human body as seen in the polymeric DDSs according tothe present invention as described above.

The hair loss preventive incorporating therein the biodegradablepolymer-based microspheres in accordance with present invention isadministered through the syringe into the skin of the patient, allowingthe hair loss preventive/therapeutic agent/drug included in themicrospheres to be released into the body in a controlled manner, as thebiodegradable polymer degrades, for an extended period of time, e.g.,one to three months, depending on the drug design requirements. Inaddition, since it is possible to mass produce monodispersebiodegradable microspheres, i.e., microspheres having a narrow particlesize distribution, through the use of the basic principles and the massproduction apparatus according to the present invention, the durationand degree of the drug release of the drug can be controlled tightly,eliminating or greatly reducing the inconvenience associated with theoral intaking of hair loss preventive/therapeutic agent/drug on adaily-basis. In addition, the basic principles and the mass productionapparatus of the present invention allow the mass production ofmonodisperse biodegradable polymer-based microspheres for use in hairloss preventives having a size close to the desired size possible with ahigh yield.

In one embodiment of the microspheres for use in hair loss preventives,wherein the microspheres include a hair loss treatment agent/drug and abiodegradable polymer, the microspheres are generally spherical with auniformly distributed hair loss treatment agent/drug and biodegradablepolymer therein. The average diameter of the microspheres is about 20 to70 μm.

In one embodiment, the hair loss treatment agent/drug of the presentinvention is finasteride or dutasteride.

In one embodiment, the microspheres of the present invention maycomprise biodegradable polymer and finasteride in a weight ratio of 3:1to 9:1, preferably 4:1. When the weight ratio of the biodegradablepolymer and the finasteride is less than 3:1, the shape-retaining forceof the biodegradable polymer may be weakened, resulting in thebiodegradable polymer and the finasteride in the microspheres beingunevenly distributed. In addition, when the weight ratio of thebiodegradable polymer and the finasteride exceeds 9:1, that is, thefinasteride content in the microspheres is small, a large amount ofmicrospheres should be administered in order for the treeatment ot beeffective, which, in turn, would cause discomfort to the patient. Morespecifically, the biodegradable polymer in the biodegradablepolymer-phase solution contains 10 to 20 wt %, preferably 15 wt %, butis not limited to the above examples.

In one embodiment, the microspheres of the present invention are capableof continuously releasing the finasteride for one to three months in thebody.

In one embodiment, the biodegradable polymer of the present invention isselected from the group consisting of polylactic acid, polylactide,polylactic-co-glycolic acid, polylactide-co-glycolide (PLGA),polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydrides,polyorthoesters, polycaprolactones, polyhydroxyvalates,polyhydroxybutyrates, polyamino acid, and combinations thereof.

In one embodiment, the microspheres of the present invention areprepared using the basic droplet-based HCMMM microsphere manufacturingmethod described above with reference to FIG. 2, wherein the mixture ofthe solvent, the hair loss preventive agent/drug and the biodegradablepolymer are properly mixed/dissolved in the biodegradable polymer-phasesolution.

In one embodiment, the microspheres of the present invention are formedusing the mass production apparatus described with reference to FIGS. 16to 31.

[Product Example]—Preparation of the Microspheres for Use in Hair LossPreventives, Including Therein Finasteride

The size of the microspheres for subcutaneous injection in human was setto be 50 μm and the size limit to be from 20 to 70 μm considering thebiodegradation period, drug delivery rate and injectability of themicrospheres, and the concentration of the biodegradable polymer andfinasteride was set as follows.

The biodegradable polymer-phase solution was prepared by dissolvingpolylactide-co-glycolide (PLGA) and finasteride in dichloromethane. Atthis time, the concentration of polylactide-co-glycolide in thebiodegradable polymer-phase solution is 15 wt %, and the weight ratio ofpolylactide-co-glycolide and finasteride is set at 4:1.

The water-phase solution was prepared by dissolving polyvinyl alcohol(PVA) as the surfactant in pure water at a concentration of 0.25 wt %.In the mass production apparatus, one side of the microchannels, i.e.,the width or the depth or both, in the multichannel microsphere formingunit 100 was set to be 50 μm, in accordance with the basic principlesdeveloped.

The water-phase solution and biodegradable polymer-phase solution wereintroduced into the microchannels formed on a silicon wafer. The flowrate of the water-phase solution was adjusted while holding the flowrate of the biodegradable polymer-phase solution constant until thediameter of the microspheres formed reaches the target diameter, i.e.,50 μm.

Then, the size of the microspheres formed was fine-tuned by adjustingthe flow rate of the biodegradable polymer phase solution while the flowrate of the aqueous solution was kept constant.

Referring to FIG. 16, the flow rate of water-phase solution and the flowrate of the biodegradable polymer-phase solution in the multichannelforming unit 100 are adjusted using the flow rate control unit 200.

The temperature of the water-phase solution and the biodegradablepolymer-phase solution were maintained at 15° C.

The microspheres in the product reservoir 600 (see FIG. 16) werecollected. Thereafter, the solvent, e.g., dichloromethane, was firstextracted from the collected microspheres. After filtering thewater-phase solution containing the microspheres, the residualsurfactant and solvent, e.g., PVA and dichloromethane, were removed fromthe microspheres through washing before being dried, resulting in theformation of the desired monodisperse biodegradable polymer-basedmicrospheres. The microspheres formed in the product reservoir 600 (seeFIG. 16) were collected to be stirred. There are three stirring stages.The first stage stirring was carried out at a speed of 1000 rpm for 1and half hours at 15° C., followed by the second stage stirring, at atemperature of to 20° C., at a speed of 1000 rpm for 1 hour and finally,the third stage stirring, a temperature of 25° C. at a speed of 1000 rpmfor 1 hour. The desired microspheres were obtained by washing severaltimes the microspheres that underwent the three stirring stages withpurified water and by lyophilizing the microspheres that underwent thewashing stages.

[Comparative Example]—Oral Hair Loss Treatment Agent/Drug

7.5 mg of hydroxypropyl beta cyclodextrin was dissolved in 100 mg ofethanol. 5 mg of finasteride was dissolved in a mixed solution in whichhydroxypropyl beta cyclodextrin was dissolved in ethanol and dried at40° C. for 12 hours. After drying, the dried material was sieved using ascreen (30 mesh), 172.5 mg of microcrystalline cellulose was added andmixed to prepare a mixture. 5 mg of magnesium stearate was added to themixture, and the mixture was then tabulated to prepare the treatmentagent/drug in an oral dosage form.

CONCLUSION

As with other microparticle engineering technologies, however flexiblethe methodology may be, it must still aim to contend with existinglarge-scale manufacturing processes, and the HCMMM processing platformand method described hereinabove can handle the issues of scale-up andindustrial-scale manufacture in unique way. The HCMMM processingplatform of the present invention can produce monodisperse microspheresin precision engineered, microfluidic circuits. Working alone, a singlefluidic circuit may produce only small volumes of particles. Yet withintelligent fluid distribution and handling described in the presentinvention, it may become possible to integrate the single fluid circuitsto produce massively parallel arrays of circuits. The HCMMM processingplatform and method of the present invention make it possible to massproduce monodisperse microspheres with an overall product sizedistribution of usually less than 1% in a considerably less space and atconsiderably lesser cost, as compared to conventional processes usedtherefor.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications and patentdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication or patent document were so individually denoted.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A multi-channel microsphere forming device forforming microspheres from a first source material and a second sourcematerial immiscible with the first source material, the devicecomprising: an upper case including a first annular manifold formed on aside of the upper case, a second annular manifold radially inside of thefirst annular manifold on the side of the upper case, a first inlet lineconfigured to deliver the first source material and the second inletline configured to deliver the second source material to the secondannular manifold, a second annular manifold formed on the side of theupper case radially inside of the first annular manifold; a lower caseincluding a product exhausting hole formed at a center of the lowercase; a lower multi-channel plate disposed on the lower case andincluding a plurality of first microchannels radially arranged andformed on a side of the lower multi-channel plate, a plurality of secondmicrochannels radially arranged and formed on a side of the lowermulti-channel plate, a plurality of third microchannels radiallyarranged and formed on a side of the lower multi-channel plate, and acenter through-hole formed at a center of the lower multi-channel plate,wherein the plurality of first microchannels and the plurality of secondmicrochannels are merged at a plurality of first merging points and theplurality of third microchannels are arranged in direction to centerthrough-hole from the plurality of first merging points; and an uppermulti-channel plate disposed between the upper case and the lowermulti-channel plate, including a plurality of first channel connectionholes disposed between the plurality of first microchannels and thefirst annular manifold, and a plurality of second channel connectionholes disposed between the first annular manifold and the plurality ofsecond microchannels.
 2. The device of claim 1, further comprising aplurality of O-rings disposed between the upper case and the uppermulti-channel plate, wherein the plurality of O-rings include at leastone first O-ring disposed radially inwardly or outwardly adjacent to thefirst annular manifold and at least one second O-ring disposed radiallyinwardly or outwardly adjacent to the second annular manifold.
 3. Thedevice of claim 1, wherein the first annular manifold and the secondannular manifold are annular recess structures formed on the side of theupper case, and wherein the first annular manifold and the secondannular manifold has larger volumes than the plurality of firstmicrochannels, the plurality of second microchannels and the pluralityof third microchannels to uniformly and consistently distribute thefirst source material or the second source material to the plurality offirst microchannels or the plurality of second microchannels and secondmicrochannels, wherein the first inlet line and the second inlet lineare respectively in fluid communication with the first annular manifoldand the second annular manifold through the upper case from another sideof the upper case, wherein the plurality of first channel connectionholes are arranged along a first circle having a first diameter, and theplurality of second channel connection holes are arranged along a secondcircle having a second diameter smaller than the first diameter, andwherein the plurality of first channel connection holes and theplurality of second channel connection holes are arranged coaxially. 4.The device of claim 1, wherein the plurality of first microchannels andthe plurality of second microchannels are arranged radially, and whereina plurality of flow paths extending from the plurality of first channelconnection holes via the plurality of first microchannels and theplurality of third microchannels to the center through-hole and aplurality of flow paths extending from the plurality of second channelconnection holes via the plurality of second microchannels and theplurality of third microchannels to the center through-hole have thesubstantially same flow length.
 5. The device of claim 1, furthercomprising a product exhausting port attached to the lower case and influid communication with the product exhausting hole, wherein theproduct exhausting port includes a coupling body and a productexhausting pipe fixed to the coupling body and fluidly connected to theproduct exhausting hole of the lower case.
 6. The device of claim 1,wherein the lower case further includes a plate seating groove formed ona side of the upper case, and the upper multi-channel plate and thelower multi-channel plate are disposed in the plate seating groove, andwherein the upper multi-channel plate further comprises an upper platealignment portion, the lower multi-channel plate further comprises alower plate alignment portion, and the plate seating grove furthercomprises case alignment portion to which the upper plate alignmentportion and the second plate alignment portion are fitted.
 7. The deviceof claim 1, wherein the upper case further comprises a third annularmanifold formed radially inward of the second annular manifold and athird inlet line delivering a third source material to the third annularmanifold, wherein the lower multi-channel plate further comprises aplurality of fourth microchannels merging with the plurality of thirdmicrochannels at a plurality of second merging points and a plurality offifth microchannels extending from the plurality of second mergingpoints to the center through-hole, and wherein the upper multi-channelplate further comprises a plurality of third channel connection holesdisposed between the plurality of fourth microchannels and the thirdannular manifold.
 8. The device of claim 1, further comprising a visionmonitoring unit mounted on the upper case, the upper case furthercomprises a monitoring opening formed at the center of the upper case,and the vision monitoring unit includes a camera imaging themicrospheres formed in the third microchannels through the monitoringopening, wherein the upper multi-channel plate is made of a glass wafer,and the lower multi-channel plate is made of a silicon wafer.
 9. Thedevice of claim 1, wherein the width or height of the plurality of thirdmicrochannels has a difference of less than 30% from the diameter of theformed microspheres.
 10. An apparatus for a mass production ofmicrospheres comprising: the multi-channel microsphere forming device ofclaim 1; a first source material reservoir containing the first sourcematerial; a second source material reservoir containing the secondsource material; and a product reservoir for accommodating themicrospheres formed in the multi-channel microsphere production device.